Antiviral jak inhibitors useful in treating or preventing retroviral and other viral infections

ABSTRACT

Compounds, compositions, and methods of treatment and prevention of HTV infection are disclosed. The compounds are pyrrolo[2,3-b]pyridines and pyrrolo[2,3-b]pyrimidine JAK inhibitors. Combinations of these JAK inhibitors and additional antiretroviral compounds, such as NRTI, NNRTI, integrase inhibitors, entry inhibitors, protease inhibitors, and the like, are also disclosed. In one embodiment, the combinations include a combination of adenine, cytosine, thymidine, and guanine nucleoside antiviral agents, optionally in further combination with at least one additional antiviral agent that works via a different mechanism than a nucleoside analog. This combination has the potential to eliminate the presence of HIV in an infected patient.

This application is a U.S. continuation-in-part application under theprovisions of 35 U.S.C. § 120 of U.S. patent application Ser. No.15/594,796 filed May 15, 2017, which is a continuation of U.S. patentapplication Ser. No. 14/808,860 filed Jul. 24, 2015, which is acontinuation of U.S. patent application Ser. No. 14/360,905 filed May27, 2014, which claims priority to International Patent Application No.PCT/US12/67369 filed Nov. 30, 2012, which in turn claims priority toU.S. Provisional Patent Application No. 61/564,994 filed Nov. 30, 2011and U.S. Provisional Patent Application No. 61/570,813 filed Dec. 15,2011. The disclosures of U.S. patent application Ser. No. 15/594,796,U.S. patent application Ser. No. 14/808,860, U.S. patent applicationSer. No. 14/360,905, International Patent Application No.PCT/US12/67369, U.S. Provisional Patent Application No. 61/564,994, andU.S. Provisional Patent Application No. 61/570,813 the contents of whichare hereby incorporated herein by reference in their respectiveentireties, for all purposes.

BACKGROUND OF THE INVENTION

In 1983, the etiological cause of AIDS was determined to be the humanimmunodeficiency virus (HIV-1). In 1985, it was reported that thesynthetic nucleoside 3′-azido-3′-deoxythymidine (AZT) inhibited thereplication of human immunodeficiency virus. Since then, a number ofother synthetic nucleosides, including 2′,3′-dideoxyinosine (DDI),2′,3′-dideoxycytidine (DDC), 2′,3′-dideoxy-2′,3′-didehydrothymidine(D4T),((1S,4R)-4-[2-amino-6-(cyclopropylamino)-9H-purin-9-yl]-2-cyclopentene-1-methanolsulfate (ABC),cis-2-hydroxymethyl-5-(5-fluorocytosin-1-yl)-1,3-oxathiolane ((−)-FTC),and (−)-cis-2-hydroxymethyl-5-(cytosin-1-yl)-1,3-oxathiolane (3TC), havebeen proven to be effective against HIV-1. After cellularphosphorylation to the 5′-triphosphate by cellular kinases, thesesynthetic nucleosides are incorporated into a growing strand of viralDNA, causing chain termination due to the absence of the 3′-hydroxylgroup. They can also inhibit the viral enzyme reverse transcriptase.

Drug-resistant variants of HIV-1 can emerge after prolonged treatmentwith an antiviral agent. Drug resistance most typically occurs bymutation of a gene that encodes for an enzyme used in viral replication,and most typically in the case of HIV-1, reverse transcriptase,protease, or DNA polymerase. Recently, it has been demonstrated that theefficacy of a drug against HIV-1 infection can be prolonged, augmented,or restored by administering the compound in combination or alternationwith a second, and perhaps third, antiviral compound that induces adifferent mutation from that caused by the principle drug.Alternatively, the pharmacokinetics, biodistribution, or other parameterof the drug can be altered by such combination or alternation therapy.In general, combination therapy is typically preferred over alternationtherapy because it induces multiple simultaneous pressures on the virus.However, drug resistance can still emerge, and no effective cure has yetbeen identified, such that a patient can ultimately stop treatment.

Treatment for AIDS using attachment and fusion inhibitors as well asother antiviral drugs has been somewhat effective. Current clinicaltreatments for HIV-1 infections include triple drug combinations calledHighly Active Antiretroviral Therapy (“HAART”). HAART typically involvesvarious combinations of nucleoside reverse transcriptase inhibitors,non-nucleoside reverse transcriptase inhibitors, and HIV-1 proteaseinhibitors. In compliant patients, HAART is effective in reducingmortality and progression of HIV-1 infection to AIDS. However, thesemultidrug therapies do not eliminate HIV-1 and long-term treatment oftenresults in multidrug resistance. Also, many of these drugs are highlytoxic and/or require complicated dosing schedules that reduce complianceand limit efficacy. There is, therefore, a continuing need for thedevelopment of additional drugs for the prevention and treatment ofHIV-1 infection and AIDS.

It would be useful to have combination therapy that minimizes thevirological failure of patients taking conventional antiretroviraltherapy. It would also be useful to provide a therapy that can provide acure for HTV/AIDS, by destroying the virus altogether in all itsreservoirs. The present invention provides such therapy, as well asmethods of treatment using the therapy.

SUMMARY OF THE INVENTION

Antiretroviral JAK inhibitors, compositions including such inhibitors,and methods for their use in treating viral infections, are provided.Examples of viruses that can be treated using the compounds describedherein include HIV, including HIV-1 and HIV-2, Flaviviridae viruses,such as HCV and Dengue, and Alphaviruses such as Chikungunya virus.

Representative JAK inhibitors include those disclosed in U.S. Pat. No.7,598,257, an example of which is Ruxolitinib (Jakafi, Incyte), whichhas the structure shown below:

Representative JAK inhibitors also include those disclosed in U.S. Pat.Nos. Re 41,783; 7,842,699; 7,803,805; 7,687,507; 7,601,727; 7,569,569;7,192,963; 7,091,208; 6,890,929, 6,696,567; 6,962,993; 6,635,762;6,627,754; and 6,610,847, an example of which is Tofacitinib (Pfizer),which has the structure shown below:

and which has the chemical name 3-{(3R,4R)-4methyl-3-[methyl-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-amino]-piperidin-1-yl}-3-oxo-propionitrile.

In one embodiment, the compounds have the formula:

wherein:

or the pharmaceutically acceptable salt thereof; wherein

R¹ is a group of the formula

wherein y is 0, 1 or 2;

R⁴ is selected from the group consisting of hydrogen, (C₁-C₆)alkyl,(C₁-C₆)alkylsulfonyl, (C₂-C₆)alkenyl, (C₂-C₆)alkynyl wherein the alkyl,alkenyl and alkynyl groups are optionally substituted by deuterium,hydroxy, amino, trifluoromethyl, (C₁-C₄)alkoxy, (C₁-C₆)acyloxy,(C₁-C₆)alkylamino, ((C₁-C₆)alkyl)₂amino, cyano, nitro, (C₂-C₆)alkenyl,(C₂-C₆)alkynyl or (C₁-C₆)acylamino; or

R⁴ is (C₃-C₁₀)cycloalkyl wherein the cycloalkyl group is optionallysubstituted by deuterium, hydroxy, amino, trifluoromethyl,(C₁-C₆)acyloxy, (C₁-C₆)acylamino, (C₁-C₆)alkylamino,((C₁-C₆)alkyl)₂amino, cyano, cyano(C₁-C₆)alkyl,trifluoromethyl(C₁-C₆)alkyl, nitro, nitro(C₁-C₆)alkyl or(C₁-C₆)acylamino;

R⁵ is (C₂-C₉)heterocycloalkyl wherein the heterocycloalkyl groups mustbe substituted by one to five carboxy, cyano, amino, deuterium, hydroxy,(C₁-C₆)alkyl, (C₁-C₆)alkoxy, halo, (C₁-C₆)acyl, (C₁-C₆)alkylamino,amino(C₁-C₆)alkyl, (C₁-C₆)alkoxy-CO—NH, (C₁-C₆)alkylamino-CO—,(C₂-C₆)alkenyl, (C₂-C₆) alkynyl, (C₁-C₆)alkylamino, amino(C₁-C₆)alkyl,hydroxy(C₁-C₆)alkyl, (C₁-C₆)alkoxy(C₁-C₆)alkyl,(C₁-C₆)acyloxy(C₁-C₆)alkyl, nitro, cyano(C₁-C₆)alkyl, halo(C₁-C₆)alkyl,nitro(C₁-C₆)alkyl, trifluoromethyl, trifluoromethyl(C₁-C₆)alkyl,(C₁-C₆)acylamino, (C₁-C₆)acylamino(C₁-C₆)alkyl,(C₁-C₆)alkoxy(C₁-C₆)acylamino, amino(C₁-C₆)acyl,amino(C₁-C₆)acyl(C₁-C₆)alkyl, (C₁-C₆)alkylamino(C₁-C₆)acyl,((C₁-C₆)alkyl)₂amino(C₁-C₆)acyl, R¹⁵R¹⁶N—CO—O—, R¹⁵R¹⁶N—CO—(C₁-C₆)alkyl,(C₁-C₆)alkyl-S(O)_(m), R¹⁵R¹⁶NS(O)_(m), R¹⁵R¹⁶NS(O)_(m)(C₁-C₆)alkyl,R¹S(O)_(m)R¹⁶N, R¹⁵S(O)_(m)R¹⁶(C₁-C₆)alkyl wherein m is 0, 1 or 2 and R⁵and R¹⁶ are each independently selected from hydrogen or (C₁-C₆)alkyl;or a group of the formula

wherein a is 0, 1, 2, 3 or 4;

b, c, e, f and g are each independently 0 or 1;

d is 0, 1, 2, or 3;

X is S(O)_(n) wherein n is 0, 1 or 2; oxygen, carbonyl or —C(═N-cyano)-;

Y is S(O)_(n) wherein n is 0, 1 or 2; or carbonyl; and

Z is carbonyl, C(O)O—, C(O)NR— or S(O)_(n) wherein n is 0, 1 or 2;

R⁶, R⁷, R⁸, R⁹, R¹⁰ and R¹¹ are each independently selected from thegroup consisting of hydrogen or (C₁-C₆)alkyl optionally substituted bydeuterium, hydroxy, amino, trifluoromethyl, (C₁-C₆)acyloxy,(C₁-C₆)acylamino, (C₁-C₆)alkylamino, ((C₁-C₆)alkyl)₂amino, cyano,cyano(C₁-C₆)alkyl, trifluoromethyl(C₁-C₆)alkyl, nitro, nitro(C₁-C₆)alkylor (C₁-C₆)acylamino;

R¹² is carboxy, cyano, amino, oxo, deuterium, hydroxy, trifluoromethyl,(C₁-C₆)alkyl, trifluoromethyl(C₁-C₆)alkyl, (C₁-C₆)alkoxy, halo,(C₁-C₆)acyl, (C₁-C₆)alkylamino, ((C₁-C₆)alkyl)₂amino, amino(C₁-C₆)alkyl,(C₁-C₆)alkoxy-CO—NH, (C₁-C₆)alkylamino-CO—, (C₂-C₆)alkenyl, (C₂-C₆)alkynyl, (C₁-C₆)alkylamino, hydroxy(C₁-C₆)alkyl,(C₁-C₆)alkoxy(C₁-C₆)alkyl, (C₁-C₆)acyloxy(C₁-C₆)alkyl, nitro,cyano(C₁-C₆)alkyl, halo(C₁-C₆)alkyl, nitro(C₁-C₆)alkyl, trifluoromethyl,trifluoromethyl(C₁-C₆)alkyl, (C₁-C₆)acylamino,(C₁-C₆)acylamino(C₁-C₆)alkyl, (C₁-C₆)alkoxy(C₁-C₆)acylamino,amino(C₁-C₆)acyl, amino(C₁-C₆)acyl(C₁-C₆)alkyl,(C₁-C₆)alkylamino(C₁-C₆)acyl, ((C₁-C₆)alkyl)₂amino(C₁-C₆)acyl,R¹⁵R¹⁶N—CO—O—, R¹⁵R¹⁶N—CO—(C₁-C₆)alkyl, R¹⁵C(O)NH, R¹⁵OC(O)NH,R¹⁵NHC(O)NH, (C₁-C₆)alkyl-S(O)_(m), (C₁-C₆)alkyl-S(O)_(m)—(C₁-C₆)alkyl,R¹⁵R¹⁶NS(O)_(m), R¹⁵R¹⁶NS(O)_(m)(C₁-C₆)alkyl, R¹S(O)_(m)R¹⁶N,R¹S(O)_(m)R¹⁶N(C₁-C₆)alkyl wherein m is 0, 1 or 2 and R¹⁵ and R¹⁶ areeach independently selected from hydrogen or (C₁-C₆)alkyl;

R² and R³ are each independently selected from the group consisting ofhydrogen, deuterium, amino, halo, hydroxy, nitro, carboxy,(C₂-C₆)alkenyl, (C₂-C₆)alkynyl, trifluoromethyl, trifluoromethoxy,(C₁-C₆)alkyl, (C₁-C₆)alkoxy, (C₃-C₁₀)cycloalkyl wherein the alkyl,alkoxy or cycloalkyl groups are optionally substituted by one to threegroups selected from halo, hydroxy, carboxy, amino (C₁-C₆)alkylthio,(C₁-C₆)alkylamino, ((C₁-C₆)alkyl)₂amino, (C₅-C₉)heteroaryl,(C₂-C₉)heterocycloalkyl, (C₃-C₉)cycloalkyl or (C₆-C₁₀)aryl; or R² and R³are each independently (C₃-C₁₀)cycloalkyl, (C₃-C₁₀)cycloalkoxy,(C₁-C₆)alkylamino, ((C₁-C₆)alkyl)₂amino, (C₆-C₁₀)arylamino,(C₁-C₆)alkylthio, (C₆-C₁₀)arylthio, (C₁-C₆)alkylsulfinyl,(C₆-C₁₀)arylsulfinyl, (C₁-C₆)alkylsulfonyl, (C₆-C₁₀)arylsulfonyl,(C₁-C₆)acyl, (C₁-C₆)alkoxy-CO—NH—, (C₁-C₆)alkylamino-CO—,(C₁-C₉)heteroaryl, (C₂-C₉)heterocycloalkyl or (C₆-C₁₀)aryl wherein theheteroaryl, heterocycloalkyl and aryl groups are optionally substitutedby one to three halo, (C₁-C₆)alkyl, (C₁-C₆)alkyl-CO—NH—,(C₁-C₆)alkoxy-CO—NH—, (C₁-C₆)alkyl-CO—NH—(C₁-C₆)alkyl,(C₁-C₆)alkoxy-CO—NH—(C₁-C₆)alkyl, (C₁-C₆)alkoxy-CO—NH—(C₁-C₆)alkoxy,carboxy, carboxy(C₁-C₆)alkyl, carboxy(C₁-C₆)alkoxy,benzyloxycarbonyl(C₁-C₆)alkoxy, (C₁-C₆)alkoxycarbonyl(C₁-C₆)alkoxy,(C₆-C₁₀)aryl, amino, amino(C₁-C₆)alkyl, (C₁-C₆)alkoxycarbonylamino,(C₆-C₁₀)aryl(C₁-C₆)alkoxycarbonylamino, (C₁-C₆)alkylamino,((C₁-C₆)alkyl)₂amino, (C₁-C₆)alkylamino(C₁-C₆)alkyl,((C₁-C₆)alkyl)₂amino(C₁-C₆)alkyl, hydroxy, (C₁-C₆)alkoxy, carboxy,carboxy(C₁-C₆)alkyl, (C₁-C₆)alkoxycarbonyl,(C₁-C₆)alkoxycarbonyl(C₁-C₆)alkyl, (C₁-C₆)alkoxy-CO—NH—,(C₁-C₆)alkyl-CO—NH—, cyano, (C₅-C₉)heterocycloalkyl, amino-CO—NH—,(C₁-C₆)alkylamino-CO—NH—, ((C₁-C₆)alkyl)₂amino-CO—NH—,(C₆-C₁₀)arylamino-CO—NH—, (C₅-C₉)heteroarylamino-CO—NH—,(C₁-C₆)alkylamino-CO—NH—(C₁-C₆)alkyl,((C₁-C₆)alkyl)₂amino-CO—NH—(C₁-C₆)alkyl,(C₆-C₁₀)arylamino-CO—NH—(C₁-C₆)alkyl,(C₅-C₉)heteroarylamino-CO—NH—(C₁-C₆)alkyl, (C₁-C₆)alkylsulfonyl,(C₁-C₆)alkylsulfonylamino, (C₁-C₆)alkylsulfonylamino(C₁-C₆)alkyl,(C₆-C₁₀)arylsulfonyl, (C₆-C₁₀)arylsulfonylamino,(C₆-C₁₀)arylsulfonylamino(C₁-C₆)alkyl, (C₁-C₆)alkylsulfonylamino,(C₁-C₆)alkylsulfonylamino(C₁-C₆)alkyl, (C₅-C₉)heteroaryl or(C₂-C₉)heterocycloalkyl.

The JAK inhibitors also include compounds of Formula B:

including pharmaceutically acceptable salt forms or prodrugs thereof,wherein:

A¹ and A² are independently selected from C and N;

T, U, and V are independently selected from O, S, N, CR⁵, and NR⁶;

wherein the 5-membered ring formed by A¹, A², U, T, and V is aromatic;

X is N or CR⁴;

Y is C₁₋₈ alkylene, C₂₋₈ alkenylene, C₂₋₈ alkynylene,(CR¹¹R¹²)_(p)—(C₃₋₁₀ cycloalkylene)-(CR¹¹R¹²)_(q),(CR¹¹R¹²)_(p)-(arylene)-(CR¹¹R¹²)_(q), (CR¹¹R¹²)_(\p), —(C₁₋₁₀heterocycloalkylene)-(CR¹¹R¹²)_(q),(CR¹¹R¹²)_(p)-(heteroarylene)-(CR¹¹R¹²)_(q),(CR¹¹R¹²)_(p)O(CR¹¹R¹²)_(q), (CR¹¹R¹²)_(p)S(CR¹¹R¹²)_(q),(CR¹¹R¹²)_(p)C(O)(CR¹¹R¹²)_(q), (CR¹¹R¹²)_(p)C(O)NR_(c)(CR¹¹R¹²)_(q),(CR¹¹R¹²)_(p)C(O)O(CR¹¹R¹²)_(q), (CR¹¹R¹²)_(p)OC(O)(CR¹¹R¹²)_(q),(CR¹¹R¹²)_(p)OC(O)NR_(c)(CR¹¹R¹²)_(q), (CR¹¹R¹²)_(p)NR(CR¹¹R¹²)_(q),(CR¹¹R¹²)_(p)NR^(c)C(O)NR^(d)(CR¹¹R¹²)_(q),(CR¹¹R¹²)_(p)S(O)(CR¹¹R¹²)_(q), (CR¹¹R¹²)_(p)S(O)NR^(c)(CR¹¹R¹²)_(q),(CR¹¹R¹²)_(p)S(O)₂(CR¹¹R¹²)_(q), or(CR¹¹R¹²)_(p)S(O)₂NR^(c)(CR¹¹R¹²)_(q), wherein said C₁₋₈ alkylene, C₂₋₈alkenylene, C₂₋₈ alkynylene, cycloalkylene, arylene,heterocycloalkylene, or heteroarylene, is optionally substituted with 1,2, or 3 substituents independently selected from -D¹-D²-D³-D⁴;

Z is H, halo, C₁₋₄ alkyl, C₂₋₄ alkenyl, C₂₋₄ alkynyl, C₁₋₄ haloalkyl,halosulfanyl, C₁₋₄ hydroxyalkyl, C₁₋₄ cyanoalkyl, ═C—R^(i), ═N—R^(i),Cy¹, CN, NO₂, OR^(a), SR^(a), C(O)R^(b), C(O)NR^(c)R^(d), C(O)OR^(a),OC(O)R^(b), OC(O)NR^(c)R^(d), NR^(c)R^(d), NR^(c)C(O)R^(b),NR^(c)C(O)NR^(c)R^(d), NR^(c)C(O)OR^(a), C(═NR^(i))NR^(c)R^(d),NR^(c)C(═NR^(i))NR^(c)R^(d), S(O)R^(b), S(O)NR^(c)R^(d), S(O)₂R^(b),NR^(c)S(O)₂R^(b), C(═NOH)R^(b), C(═NO(C₁₋₆alkyl)R^(b), andS(O)₂NR^(c)R^(d), wherein said C₁₋₈ alkyl, C₂₋₈ alkenyl, or C₂₋₈alkynyl, is optionally substituted with 1, 2, 3, 4, 5, or 6 substituentsindependently selected from halo, C₁₋₄ alkyl, C₂₋₄ alkenyl, C₂₋₄alkynyl, C₁₋₄ haloalkyl, halosulfanyl, C₁₋₄ hydroxyalkyl, C₁₋₄cyanoalkyl, Cy¹, CN, N₂, OR^(a), SR^(a), C(O)R^(b), C(O)NR^(c)R^(d),C(O)OR^(a), OC(O)R^(b), OC(O)NR^(c)R^(d), NR^(c)R^(d), NR^(c)C(O)R^(b),NR^(c)C(O)NR^(c)R^(d), NR^(c)C(O)OR^(a), C(═NR^(i))NR^(c)R^(d),NR^(c)C(═NR^(i))NR^(c)R^(d), S(O)R^(b), S(O)NR^(c)R^(d), S(O)₂R^(b),NR^(c)S(O)₂R^(b), C(═NOH)R^(b), C(═NO(C₁₋₆ alkyl))R^(b), andS(O)₂NR^(c)R^(d);

wherein when Z is H, n is 1;

or the —(Y)_(n)—Z moiety is taken together with i) A² to which themoiety is attached, ii) R⁵ or R⁶ of either T or V, and iii) the C or Natom to which the R⁵ or R⁶ of either T or V is attached to form a 4- to20-membered aryl, cycloalkyl, heteroaryl, or heterocycloalkyl ring fusedto the 5-membered ring formed by A¹ A², U, T, and V, wherein said 4- to20-membered aryl, cycloalkyl, heteroaryl, or heterocycloalkyl ring isoptionally substituted by 1, 2, 3, 4, or 5 substitutes independentlyselected from —(W)_(m)-Q;

W is C₁₋₈ alkylenyl, C₂₋₈ alkenylenyl, C₂₋₈ alkynylenyl, O, S, C(O),C(O)NR^(c′), C(O)O, OC(O), OC(O)NR^(c′), NR^(c′), NR^(c′)C(O)NR^(d′),S(O), S(O)NR^(c′), S(O)₂, or S(O)₂NR^(c′);

Q is H, halo, CN, NO₂, C₁₋₈ alkyl, C₂₋₈ alkenyl, C₂₋₈ alkynyl, C₁₋₈haloalkyl, halosulfanyl, aryl, cycloalkyl, heteroaryl, orheterocycloalkyl, wherein said C₁₋₈ alkyl, C₂₋₈ alkenyl, C₂₋₄ alkynyl,C₁₋₈ haloalkyl, aryl, cycloalkyl, heteroaryl, or heterocycloalkyl isoptionally substituted with 1, 2, 3 or 4 substituents independentlyselected from halo, C₁₋₄ alkyl, C₂₋₄ alkenyl, C₂₋₄ alkynyl, C₁₋₄haloalkyl, halosulfanyl, C₁₋₄ hydroxyalkyl, C₁₋₄ cyanoalkyl, Cy², CN,NO₂, OR^(a′), SR^(a′), C(O)R^(b′), C(O)NR^(c′)R^(d′), C(O)OR^(a′),OC(O)R^(b′), OC(O)NR^(c′)R^(d′), NR^(c′)R^(d′), NR^(c′)C(O)R^(b′),NR^(c′)C(O)NR^(c′)R^(d′), N R^(c′)C(O)OR^(a′), S(O)R^(b′),S(O)NR^(c′)R^(d′), S(O)₂R^(b′), NR^(c)′S(O)₂R^(b′), andS(O)₂NR^(c′)R^(d′);

Cy¹ and Cy² are independently selected from aryl, heteroaryl,cycloalkyl, and heterocycloalkyl, each optionally substituted by 1, 2,3, 4 or 5 substituents independently selected from halo, C₁₋₄ alkyl,C₂₋₄ alkenyl, C₂₋₄ alkynyl, C₁₋₄ haloalkyl, halosulfanyl, C₁₋₄hydroxyalkyl, C₁₋₄ cyanoalkyl, CN, NO₂, OR^(a″), SR^(a″), C(O)R^(b)″,C(O)NR^(c′)′R^(d″), C(O)OR^(a″), OC(O)R^(b″), OC(O)N R^(c′)′R^(d″),NR^(c′)′R^(d″), NR^(c′)′C(O)R^(b″), NR^(c″)C(O)OR^(a″),NR^(c″)S(O)R^(b″), NR^(c″)S(O)₂R^(b″), S(O)R^(b″), S(O)NR^(c″)R^(d″),S(O)₂R^(b″), and S(O)₂NR^(c″)R^(d″);

R¹, R², R³, and R⁴ are independently selected from H, halo, C₁₋₄ alkyl,C₂₋₄ alkenyl, C₂₋₄ alkynyl, C₁₋₄ haloalkyl, halosulfanyl, aryl,cycloalkyl, heteroaryl, heterocycloalkyl, CN, NO₂, OR⁷, SR⁷, C(O)R⁸,C(O)NR⁹R¹⁰, C(O)OR⁷OC(O)R⁸, OC(O)NR⁹R¹⁰, NR⁹R¹⁰, NR⁹C(O)R⁸,NR^(c)C(O)OR⁷, S(O)R⁸, S(O)NR⁹R¹⁰, S(O)₂R⁸, NR⁹S(O)₂R⁸, and S(O)₂NR⁹R¹⁰;

R⁵ is H, halo, C₁₋₄ alkyl, C₂₋₄ alkenyl, C₂₋₄ alkynyl, C₁₋₄ haloalkyl,halosulfanyl, CN, NO₂, OR⁷, SR⁷, C(O)R⁸, C(O)NR⁹R¹⁰, C(O)OR⁷, OC(O)R⁸,OC(O)NR⁹R¹⁰, NR⁹R¹⁰, NR⁹C(O)R⁸, NR⁹C(O)OR⁷, S(O)R⁸, S(O)NR⁹R¹⁰, S(O)₂R⁸,NR⁹S(O)₂R⁸, or S(O)₂NR⁹R¹⁰;

R⁶ is H, C₁₋₄ alkyl, C₂₋₄ alkenyl, C₂₋₄ alkynyl, C₁₋₄ haloalkyl, OR,C(O)R⁸, C(O)NR⁹R¹⁰, C(O)OR⁷, S(O)R⁸, S(O)NR⁹R¹⁰, S(O)₂R⁸, orS(O)₂NR⁹R¹⁰;

R⁷ is H, C₁₋₆ alkyl, C₁₋₆ haloalkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, aryl,cycloalkyl, heteroaryl, heterocycloalkyl, arylalkyl, heteroarylalkyl,cycloalkylalkyl or heterocycloalkylalkyl;

R⁸ is H, C₁₋₆ alkyl, C₁₋₆ haloalkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, aryl,cycloalkyl, heteroaryl, heterocycloalkyl, arylalkyl, heteroarylalkyl,cycloalkylalkyl or heterocycloalkylalkyl;

R⁹ and R¹⁰ are independently selected from H, C₁₋₁₀ alkyl, C₁₋₆haloalkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₁₋₆ alkylcarbonyl, arylcarbonyl,C₁₋₄ alkylsulfonyl, arylsulfonyl, aryl, heteroaryl, cycloalkyl,heterocycloalkyl, arylalkyl, heteroarylalkyl, cycloalkylalkyl andheterocycloalkylalkyl;

or R⁹ and R¹⁰ together with the N atom to which they are attached form a4-, 5-, 6- or 7-membered heterocycloalkyl group;

R¹¹ and R¹² are independently selected from H and -E¹-E²-E³-E⁴;

D¹ and E¹ are independently absent or independently selected from C₁₋₆alkylene, C₂₋₆ alkenylene, C₂₋₆ alkynylene, arylene, cycloalkylene,heteroarylene, and heterocycloalkylene, wherein each of the C₁₋₆alkylene, C₂₋₆ alkenylene, C₂₋₆ alkynylene, arylene, cycloalkylene,heteroarylene, and heterocycloalkylene is optionally substituted by 1, 2or 3 substituents independently selected from halo, CN, NO₂, N₃, SCN,OH, C₁₋₆ alkyl, C₁₋₆ haloalkyl, C₂₋₈ alkoxyalkyl, C₁₋₆ alkoxy, C₁₋₆haloalkoxy, amino, C₁₋₄ alkylamino, and C₂₋₄ dialkylamino;

D² and E² are independently absent or independently selected from C₁₋₆alkylene, C₂₋₆ alkenylene, C₂₋₆ alkynylene, (C₁₋₆ alkylene)_(r)-O—(C₁₋₆alkylene)_(s), (C₁₋₄ alkylene)_(r)-S—(C₁₋₆ alkylene)_(s), (C₁₋₆alkylene)_(r)-NR^(c)-(C₁₋₆ alkylene)_(s), (C₁₋₆ alkylene)_(r)-CO—(C₁₋₆alkylene)_(s), (C₁₋₆ alkylene)_(r)-COO—(C₁₋₆ alkylene)_(s), (C₁₋₆alkylene)_(r)-CONR^(c)-(C₁₋₆ alkylene)_(s), (C₁₋₆ alkylene)_(r)-SO—(C₁₋₄alkylene)_(s), (C₁₋₆ alkylene)_(r)-SO₂—(C₁₋₆ alkylene)_(s), (C₁₋₆alkylene)_(r)-SONR^(c)-(C₁₋₆ alkylene)_(s), and (C₁₋₄alkylene)_(r)-NR^(e)CONR^(f)—(C₁₋₆ alkylene)_(s), wherein each of theC₁₋₆ alkylene, C₂₋₆ alkenylene, and C₂₋₆ alkynylene is optionallysubstituted by 1, 2 or 3 substituents independently selected from halo,CN, NO₂, N₃, SCN, OH, C₁₋₆ alkyl, C₁₋₆ haloalkyl, C₂₋₈ alkoxyalkyl, C₁₋₆alkoxy, C₁₋₆ haloalkoxy, amino, C₁₋₆ alkylamino, and C₂₋₈ dialkylamino;

D³ and E³ are independently absent or independently selected from C₁₋₆alkylene, C₂₋₆ alkenylene, C₂₋₆ alkynylene, arylene, cycloalkylene,heteroarylene, and heterocycloalkylene, wherein each of the C₁₋₆alkylene, C₂₋₆ alkenylene, C₂₋₆ alkynylene, arylene, cycloalkylene,heteroarylene, and heterocycloalkylene is optionally substituted by 1, 2or 3 substituents independently selected from halo, CN, NO₂, N₃, SCN,OH, C₁₋₆ alkyl, C₁₋₆ haloalkyl, C₂₋₈ alkoxyalkyl, C₁₋₆ alkoxy, C₁₋₆haloalkoxy, amino, C₁₋₆ alkylamino, and C₂₋₈ dialkylamino;

E⁴ and E⁴ are independently selected from H, halo, C₁₋₄ alkyl, C₂₋₄alkenyl, C₂₋₄ alkynyl, C₁₋₄ haloalkyl, halosulfanyl, C₁₋₄ hydroxyalkyl,C₁₋₄ cyanoalkyl, Cy¹, CN, NO₂, OR^(a), SR^(a), C(O)R^(b),C(O)NR^(c)R^(a), C(O)OR^(a), OC(O)R^(b)OC(O)NR^(c)R^(d) NR^(c)R^(d),NR^(c)C(O)R, NR^(c)C(O)NR^(c)R^(d), NR^(c)C(O)OR^(a),C(═NR^(i))NR^(c)R^(d), NR^(c)C(═NR^(i))NR^(c)R^(d), S(O)R^(b),S(O)NR^(c)R^(d), S(O)₂Rb, NR^(c)S(O)₂R^(b), C(═NOH)R^(b), C(═NO(C₁₋₄alkyl)R^(b), and S(O)₂NR^(c)R^(d), wherein said C₁₋₈ alkyl, C₂₋₈alkenyl, or C₂₋₈ alkynyl, is optionally substituted with 1, 2, 3, 4, 5,or 6 substituents independently selected from halo, C₁₋₄ alkyl, C₂₋₄alkenyl, C₂₋₄ alkynyl, C₁₋₄ haloalkyl, halosulfanyl, C₁₋₄ hydroxyalkyl,C₁₋₄ cyanoalkyl, Cy¹, CN, NO₂, OR^(a), SR^(a), C(O)R^(b),C(O)NR^(c)R^(d), C(O)OR^(a), OC(O)R^(b), OC(O)NR^(c)R^(d), NR^(c)R^(d),NR^(c)C(O)R^(b), NR^(c)C(O)NR^(c)R^(d), NR^(c)C(O)OR^(a),C(═NR^(i))NR^(c)R^(d), NR^(c)C(═NR^(i))NR^(c)R^(d), S(O)R^(b),S(O)NR^(c)R^(d), S(O)₂R^(b), NR^(c)S(O)₂R^(b), C(═NOH)R^(b), C(═NO(C₁₋₆alkyl))R^(b), and S(O)₂NR^(c)R^(d);

R^(a) is H, Cy¹, —(C₁₋₆ alkyl)-Cy¹, C₁₋₆ alkyl, C₁₋₆ haloalkyl, C₂₋₆alkenyl, C₂₋₆ alkynyl, wherein said C₁₋₆ alkyl, C₁₋₆ haloalkyl, C₂₋₆alkenyl, or C₂₋₆ alkynyl is optionally substituted with 1, 2, or 3substituents independently selected from OH, CN, amino, halo, C₁₋₆alkyl, C₁₋₆ haloalkyl, halosulfanyl, aryl, arylalkyl, heteroaryl,heteroarylalkyl, cycloalkyl and heterocycloalkyl;

R^(b) is H, Cy¹, —(C₁₋₆ alkyl)-Cy¹, C₁₋₆ alkyl, C₁₋₆ haloalkyl, C₂₋₆alkenyl, C₂₋₆ alkynyl, wherein said C₁₋₆ alkyl, C₁₋₆ haloalkyl, C₂₋₆alkenyl, or C₂₋₆ alkynyl is optionally substituted with 1, 2, or 3substituents independently selected from OH, CN, amino, halo, C₁₋₆alkyl, C₁₋₆ haloalkyl, C₁₋₆ haloalkyl, halosulfanyl, aryl, arylalkyl,heteroaryl, heteroarylalkyl, cycloalkyl and heterocycloalkyl;

R^(a)′ and R^(a)″ are independently selected from H, C₁₋₆ alkyl, C₁₋₆haloalkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, aryl, cycloalkyl, heteroaryl,heterocycloalkyl, arylalkyl, heteroarylalkyl, cycloalkylalkyl andheterocycloalkylalkyl, wherein said C₁₋₆ alkyl, C₁₋₆ haloalkyl, C₂₋₆alkenyl, C₂₋₆ alkynyl, aryl, cycloalkyl, heteroaryl, heterocycloalkyl,arylalkyl, heteroarylalkyl, cycloalkylalkyl or heterocycloalkylalkyl isoptionally substituted with 1, 2, or 3 substituents independentlyselected from OH, CN, amino, halo, C₁₋₆ alkyl, C₁₋₆ haloalkyl,halosulfanyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, cycloalkyland heterocycloalkyl;

R^(b′) and R^(b″) are independently selected from H, C₁₋₆ alkyl, C₁₋₆haloalkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, aryl, cycloalkyl, heteroaryl,heterocycloalkyl, arylalkyl, heteroarylalkyl, cycloalkylalkyl andheterocycloalkylalkyl, wherein said C₁₋₆ alkyl, C₁₋₆ haloalkyl, C₂₋₆alkenyl, C₂₋₆ alkynyl, aryl, cycloalkyl, heteroaryl, heterocycloalkyl,arylalkyl, heteroarylalkyl, cycloalkylalkyl or heterocycloalkylalkyl isoptionally substituted with 1, 2, or 3 substituents independentlyselected from OH, CN, amino, halo, C₁₋₆ alkyl, C₁₋₆ haloalkyl, C₁₋₆haloalkyl, halosulfanyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl,cycloalkyl and heterocycloalkyl;

R^(c) and R^(d) are independently selected from H, Cy¹, —(C₁₋₆alkyl)-Cy¹, C₁₋₁₀ alkyl, C₁₋₆ haloalkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl,wherein said C₁₋₁₀ alkyl, C₁₋₆ haloalkyl, C₂₋₆ alkenyl, or C₂₋₆ alkynyl,is optionally substituted with 1, 2, or 3 substituents independentlyselected from Cy¹, —(C₁₋₆ alkyl)-Cy¹, OH, CN, amino, halo, C₁₋₆ alkyl,C₁₋₆ haloalkyl, C₁₋₆ haloalkyl, and halosulfanyl;

or R^(c) and R^(d) together with the N atom to which they are attachedform a 4-, 5-, 6- or 7-membered heterocycloalkyl group optionallysubstituted with 1, 2, or 3 substituents independently selected fromCy¹, —(C₁₋₆ alkyl)-Cy¹, OH, CN, amino, halo, C₁₋₆ alkyl, C₁₋₆ haloalkyl,C₁₋₆ haloalkyl, and halosulfanyl;

R^(c)′ and R^(d)′ are independently selected from H, C₁₋₁₀ alkyl, C₁₋₆haloalkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, aryl, heteroaryl, cycloalkyl,heterocycloalkyl, arylalkyl, heteroarylalkyl, cycloalkylalkyl andheterocycloalkylalkyl, wherein said C₁₋₁₀ alkyl, C₁₋₆ haloalkyl, C₂₋₆alkenyl, C₂₋₆ alkynyl, aryl, heteroaryl, cycloalkyl, heterocycloalkyl,arylalkyl, heteroarylalkyl, cycloalkylalkyl or heterocycloalkylalkyl isoptionally substituted with 1, 2, or 3 substituents independentlyselected from OH, CN, amino, halo, C₁₋₆ alkyl, C₁₋₆ haloalkyl, C₁₋₆haloalkyl, halosulfanyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl,cycloalkyl and heterocycloalkyl;

or R^(c)′ and R^(d)′ together with the N atom to which they are attachedform a 4-, 5-, 6- or 7-membered heterocycloalkyl group optionallysubstituted with 1, 2, or 3 substituents independently selected from OH,CN, amino, halo, C₁₋₆ alkyl, C₁₋₆ haloalkyl, C₁₋₆ haloalkyl,halosulfanyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, cycloalkyland heterocycloalkyl;

R^(c)″ and R^(d)″ are independently selected from H, C₁₋₁₀ alkyl, C₁₋₆haloalkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, aryl, heteroaryl, cycloalkyl,heterocycloalkyl, arylalkyl, heteroarylalkyl, cycloalkylalkyl andheterocycloalkylalkyl, wherein said C₁₋₁₀ alkyl, C₁₋₆ haloalkyl, C₂₋₆alkenyl, C₂₋₆ alkynyl, aryl, heteroaryl, cycloalkyl, heterocycloalkyl,arylalkyl, heteroarylalkyl, cycloalkylalkyl or heterocycloalkylalkyl isoptionally substituted with 1, 2, or 3 substituents independentlyselected from OH, CN, amino, halo, C₁₋₆ alkyl, C₁₋₆ haloalkyl,halosulfanyl, C₁₋₆ haloalkyl, aryl, arylalkyl, heteroaryl,heteroarylalkyl, cycloalkyl and heterocycloalkyl;

or R^(c)″ and R^(d)″ together with the N atom to which they are attachedform a 4-, 5-, 6- or 7-membered heterocycloalkyl group optionallysubstituted with 1, 2, or 3 substituents independently selected from OH.CN, amino, halo, C₁₋₆ alkyl, C₁₋₆ haloalkyl, C₁₋₆ haloalkyl,halosulfanyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, cycloalkyland heterocycloalkyl;

R^(i) is H, CN, NO₂, or C₁₋₆ alkyl;

R^(e) and R^(f) are independently selected from H and C₁₋₆ alkyl;

R^(i) is H, CN, or NO₂;

m is 0 or 1;

n is 0 or 1;

p is 0, 1, 2, 3, 4, 5, or 6;

q is 0, 1, 2, 3, 4, 5 or 6;

r is 0 or 1; and

s is 0 or 1.

Additional JAK inhibitors include CEP-701 (Lestaurtinib, CephalonTechnology), a JAK 2 FL3 kinase, AZD1480 (Astra Zeneca), a JAK 2inhibitor, LY3009104/INCB28050 (Eli Lilly, Incyte), a JAK 1/2 inhibitor,Pacritinib/SB1518 (S*BIO), a JAK 2 inhibitor. VX-509 (Vertex), a JAK 3inhibitor, GLPG0634 (Galapagos), a JAK 1 inhibitor, INC424 (Novartis), aJAK inhibitor, R-348 (Rigel), a JAK 3 inhibitor, CYT387 (YM Bioscience),a JAK1/2 inhibitor, TG 10138, a JAK 2 inhibitor, AEG 3482 (Axon), a JAKinhibitor, and pharmaceutically-acceptable salts and prodrugs thereof.

Lestaurtinib has the following formula:

AEG 3482 has the following formula:

TG 10138 has the following formula:

CYT387 has the following formula:

AZD148 has the following formula:

LY3009104 is believed to be(R)-3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl-3-cyclopentyl-propanenitrile

Pacritinib has the following formula:

The compounds include those described in U.S. Publication Nos.20110020469; 20110118255; 20100311743; 20100310675; 20100280026;20100160287; 20100081657; 20100081645; 20090181938; 20080032963;20070259869; and 20070249031.

The compounds also include those described in U.S. Publication Nos.20110251215; 20110224157; 20110223210; 20110207754; 20110136781;20110086835; 20110086810; 20110082159; 20100190804; 20100022522;20090318405; 20090286778; 20090233903; 20090215766; 20090197869;20090181959; 20080312259; 20080312258; 20080188500; and 20080167287;20080039457.

The compounds also include those described in U.S. Publication Nos.20100311693; 20080021013; 20060128780; 20040186157; and 20030162775.

The compounds also include those described in U.S. Publication Nos.20110245256; 20100009978; 20090098137; and 20080261973.

The compounds also include those described in U.S. Publication No.20110092499. Representative compounds include:

1. 7-iodo-N-(4-morpholinophenyl)thieno[3,2-d]pyrimidin-2-amine 2.7-(4-aminophenyl)-N-(4-morpholinophenyl)thieno[3,2-d]pyrimidin-2-amine3.N-(4-(2-(4-morpholinophenylamino)thieno[3,2-d]pyrimidin-7-yl)phenyl)acryl-amide4.7-(3-aminophenyl)-N-(4-morpholinophenyl)thieno[3,2-d]pyrimidin-2-amine5.N-(3-(2-(4-morpholinophenylamino)thieno[3,2-d]pyrimidin-7-yl)phen-yl)acrylamide7. N-(4-morpholinophenyl)thieno[3,2-d]pyrimidin-2-amine 8. methyl2-(4-morpholinophenylamino)thieno[3,2-d]pyrimidine-7-carboxylate 9.N-(4-morpholinophenyl)-5H-pyrrolo[3,2-d]pyrimidin-2-amine 10.7-(4-amino-3-methoxyphenyl)-N-(4-morpholinophenyl)thieno[3,2-d]pyrimidin-2-amine11.4-(2-(4-morpholinophenylamino)thieno[3,2-d]pyrimidin-7-yl)benzenesulfonam-ide12.N,N-dimethyl-3-(2-(4-morpholinophenylamino)thieno[3,2-d]pyrimidin-7-yl)benzenesulfonamide13.1-ethyl-3-(2-methoxy-4-(2-(4-morpholinophenylamino)thieno[3,2-d]pyrimidin-7-yl)phenyl)urea14.N-(4-(2-(4-morpholinophenylamino)thieno[3,2-d]pyrimidin-7-yl)phenyl)metha-nesulfonamide15.2-methoxy-4-(2-(4-morpholinophenylamino)thieno[3,2-d]pyrimidin-7-yl)pheno-l16.2-cyano-N-(3-(2-(4-morpholinophenylamino)thieno[3,2-d]pyrimidin-7-yl-)phenyl)acetamide17.N-(cyanomethyl)-2-(4-morpholinophenylamino)thieno[3,2-d]pyrimidine-7-carb-oxamide18.N-(3-(2-(4-morpholinophenylamino)thieno[3,2-d]pyrimidin-7-yl)phenyl)metha-nesulfonamide19.1-ethyl-3-(4-(2-(4-morpholinophenylamino)thieno[3,2-d]pyrimidin-7-yl)-2-(-trifluoromethoxy)phenyl)urea20. N-(3-nitrophenyl)-7-phenylthieno[3,2-d]pyrimidin-2-amine 21.7-iodo-N-(3-nitrophenyl)thieno[3,2-d]pyrimidin-2-amine 22.N1-(7-(2-ethylphenyl)thieno[3,2-d]pyrimidin-2-yl)benzene-1,3-diamine 25.N-tert-butyl-3-(2-(4-morpholinophenylamino)thieno[3,2-d]pyrimidin-7-yl)be-nzenesulfonamide26. N1-(7-iodothieno[3,2-d]pyrimidin-2-yl)benzene-1,3-diamine 28.7-(4-amino-3-(trifluoromethoxy)phenyl)-N-(4-morpholinophenyl)thieno[3,2-d]pyrimidin-2-amin-e29.7-(2-ethylphenyl)-N-(4-morpholinophenyl)thieno[3,2-d]pyrimidin-2-ami-ne30.N-(3-(2-(4-morpholinophenylamino)thieno[3,2-d]pyrimidin-7-yl)phenyl-)acetamide31.N-(cyanomethyl)-N-(3-(2-(4-morpholinophenylamino)thieno[3,2-d]pyrimidin-7-yl)phenyl)methanesulfonamide32.N-(cyanomethyl)-N-(4-(2-(4-morpholinophenylamino)thieno[3,2-d]pyrimidin-7-yl)phenyl)methanesulfonamide33.N-(3-(5-methyl-2-(4-morpholinophenylamino)-5H-pyrrolo[3,2-d]pyrimidin-7-y-l)phenyl)methanesulfonamide34.4-(5-methyl-2-(4-morpholinophenylamino)-5H-pyrrolo[3,2-d]pyrimidin-7-yl)b-enzenesulfonamide36.N-(4-(5-methyl-2-(4-morpholinophenylamino)-5H-pyrrolo[3,2-d]pyrimidin-7-y-l)phenyl)methanesulfonamide37. 7-iodo-N-(4-morpholinophenyl)-5H-pyrrolo[3,2-d]pyrimidin-2-amine 38.7-(2-isopropylphenyl)-N-(4-morpholinophenyl)thieno[3,2-d]pyrimidin-2-amin-e39. 7-bromo-N-(4-morpholinophenyl)thieno[3,2-d]pyrimidin-2-amine 40.N7-(2-isopropylphenyl)-N2-(4-morpholinophenyl)thieno[3,2-d]pyrimidine-2,7-diamine41.N7-(4-isopropylphenyl)-N2-(4-morpholinophenyl)thieno[3,2-d]pyrimidine-2,7-diamine42.7-(5-amino-2-methylphenyl)-N-(4-morpholinophenyl)thieno[3,2-d]pyrimidin-2-amine43.N-(cyanomethyl)-4-(2-(4-morpholinophenylamino)thieno[3,2-d]pyri-midin-7-10yl)benzamide 44.7-iodo-N-(3-morpholinophenyl)thieno[3,2-d]pyrimidin-2-amine 45.7-(4-amino-3-nitrophenyl)-N-(4-morpholinophenyl)thieno[3,2-d]pyrimidin-2-amine46.7-(2-methoxypyridin-3-yl)-N-(4-morpholinophenyl)thieno[3,2-d]pyr-imidin-2-amine47. (3-(7-iodothieno[3,2-d]pyrimidin-2-ylamino)phenyl)methanol 48.N-tert-butyl-3-(2-(3-morpholinophenylamino)thieno[3,2-d]pyrimidin-7-yl)be-nzenesulfonamide49.N-tert-butyl-3-(2-(3-(hydroxymethyl)phenylamino)thieno[3,2-d]pyrimidin-7-yl)benzenesulfonamide50.N-(4-morpholinophenyl)-7-(4-nitrophenylthio)-5H-pyrrolo[3,2-d]pyrimidin-2-amine51.N-tert-butyl-3-(2-(3,4,5-trimethoxyphenylamino)thieno[3,2-d]pyr-imidin-7-yl)benzenesulfonamide52.7-(4-amino-3-nitrophenyl)-N-(3,4-dimethoxyphenyl)thieno[3,2-d]pyrimidin-2-amine53.N-(3,4-dimethoxyphenyl)-7-(2-methoxypyridin-3-yl)thieno[3,2-d]p-yrimidin-2-amine54.N-tert-butyl-3-(2-(3,4-dimethoxyphenylamino)thieno[3,2-d]pyrimidin-7-yl)b-enzenesulfonamide55.7-(2-aminopyrimidin-5-yl)-N-(3,4-dimethoxyphenyl)thieno[3,2-d]pyrimidin-2-amine56.N-(3,4-dimethoxyphenyl)-7-(2,6-dimethoxypyridin-3-yl)thieno[3,2-d]pyrimidin-2-amine57.N-(3,4-dimethoxyphenyl)-7-(2,4-dimethoxypyrimidin-5-yl)thieno[3,2-d]pyrim-idin-2-amine58. 7-iodo-N-(4-(morpholinomethyl)phenyl)thieno[3,2-d]pyrimidin-2-amine59.N-tert-butyl-3-(2-(4-(morpholinomethyl)phenylamino)thieno[3,2-d]pyrimidin-7-yl)benzenesulfonamide60.2-cyano-N-(4-methyl-3-(2-(4-morpholinophenylamino)thieno[3,2-d]pyrimidin-7-yl)phenyl)acetamide61. ethyl3-(2-(4-morpholinophenylamino)thieno[3,2-d]pyrimidin-7-yl)benzoate 62.7-bromo-N-(4-(2-(pyrrolidin-1-yl)ethoxy)phenyl)thieno[3,2-d]pyrimidin-2-a-mine63.N-(3-(2-(4-(2-(pyrrolidin-1-yl)ethoxy)phenylamino)thieno[3,2-d]py-rimidin-7-yl)phenyl)acetamide64.N-(cyanomethyl)-3-(2-(4-morpholinophenylamino)thieno[3,2-d]pyrimidin-7-yl-)benzamide65.N-tert-butyl-3-(2-(4-morpholinophenylamino)thieno[3,2-d]pyrimidin-7-yl)be-nzamide66.N-tert-butyl-3-(2-(4-(1-ethylpiperidin-4-yloxy)phenylamino)thieno[3,2-d]p-yrimidin-7-yl)benzenesulfonamide67. tert-butyl4-(2-(4-(morpholinomethyl)phenylamino)thieno[3,2-d]pyrimidin-7-yl)-1H-pyr-azole-1-carboxylate68.7-bromo-N-(4-((4-ethylpiperazin-1-yl)methyl)phenyl)thieno[3,2-d]pyrimidin-2-amine69.N-tert-butyl-3-(2-(4-((4-ethylpiperazin-1-yl)methyl)phenylamino)thieno[3,-2-d]pyrimidin-7-yl)benzenesulfonamide70.N-(4-((4-ethylpiperazin-1-yl)methyl)phenyl)-7-(1H-pyrazol-4-yl)thieno[3,2-d]pyrimidin-2-amine71.N-(cyanomethyl)-3-(2-(4-(morpholinomethyl)phenylamino)thieno[3,2-d]pyrimi-din-7-yl)benzamide72.N-tert-butyl-3-(2-(4-(2-(pyrrolidin-1-yl)ethoxy)phenylamino)thieno[3,2-d]-pyrimidin-7-yl)benzenesulfonamide73. tert-butylpyrrolidin-1-yl)ethoxy)phenylamino)thieno[3,2-d]pyrimidin-7-yl)benzylcarb-amate74.3-(2-(4-(2-(pyrrolidin-1-yl)ethoxy)phenylamino)thieno[3,2-d]pyri-midin-7-yl)benzenesulfonamide75.7-(3-chloro-4-fluorophenyl)-N-(4-(2-(pyrrolidin-1-yl)ethoxy)phenyl)thieno-[3,2-d]pyrimidin-2-amine76. tert-butyl4-(2-(4-(1-ethylpiperidin-4-yloxy)phenylamino)thieno[3,2-d]pyrimidin-7-yl-)-1H-pyrazole-1-carboxylate77.7-(benzo[d][1,3]dioxol-5-yl)-N-(4-(morpholinomethyl)phenyl)thieno[3,2-d]p-yrimidin-2-amine78. tert-butyl5-(2-(4-(morpholinomethyl)phenylamino)thieno[3,2-d]pyrimidin-7-yl)-1H-ind-ole-1-carboxylate79.7-(2-aminopyrimidin-5-yl)-N-(4-(morpholinomethyl)phenyl)thieno[3,2-d]pyri-midin-2-amine80. tert-butyl4-(2-(4-(morpholinomethyl)phenylamino)thieno[3,2-d]pyrimidin-7-yl)-5,6-di-hydropyridine-1(2H)-carboxylate81. tert-butylmorpholinomethyl)phenylamino)thieno[3,2-d]pyrimidin-7-yl)benzylcarbamate82.N-(3-(2-(4-(morpholinomethyl)phenylamino)thieno[3,2-d]pyrimidin-7-yl)-phenyl)acetamide83.N-(4-(2-(4-(morpholinomethyl)phenylamino)thieno[3,2-d]pyrimidin-7-yl)phen-yl)acetamide84.N-(3-(2-(4-(morpholinomethyl)phenylamino)thieno[3,2-d]pyrimidin-7-yl)phen-yl)methanesulfonamide85.7-(4-(4-methylpiperazin-1-yl)phenyl)-N-(4-(morpholinomethyl)phenyl)thieno-[3,2-d]pyrimidin-2-amine86. N-30(2-methoxy-4-(2-(4-(morpholinomethyl)phenylamino)thieno[3,2-d]pyrimidin-7-yl)phenyl)acetamide87. 7-bromo-N-(3,4,5-trimethoxyphenyl)thieno[3,2-d]pyrimidin-2-amine 88.(3-(2-(3,4,5-trimethoxyphenylamino)thieno[3,2-d]pyrimidin-7-yl)phenyl)met-hanol89.(4-(2-(3,4,5-trimethoxyphenylamino)thieno[3,2-d]pyrimidin-7-yl)p-henyl)methanol90.(3-(2-(4-morpholinophenylamino)thieno[3,2-d]pyrimidin-7-yl)phenyl)methano-191.(4-(2-(4-morpholinophenylamino)thieno[3,2-d]pyrimidin-7-yl)phenyl)me-thanol92.N-(pyrrolidin-1-yl)ethoxy)phenylamino)thieno[3,2-d]pyrimidin-7-yl)benzyl)methanesulfonamide93. tert-butylmorpholinomethyl)phenylamino)thieno[3,2-d]pyrimidin-7-yl)benzylcarbamate94.N-(4-(morpholinomethyl)phenyl)-7-(3-(piperazin-1-yl)phenyl)thieno[3,2-d]pyrimidin-2-amine95.7-(6-(2-morpholinoethylamino)pyridin-3-yl)-N-(3,4,5-trimethoxyphenyl)thie-no[3,2-d]pyrimidin-2-amine96.7-(2-ethylphenyl)-N-(4-(2-(pyrrolidin-1-yl)ethoxy)phenyl)thieno[3,2-d]pyr-imidin-2-amine97.7-(4-(aminomethyl)phenyl)-N-(4-(morpholinomethyl)phenyl)thieno[3,2-d]pyri-midin-2-amine98.N-(4-(1-ethylpiperidin-4-yloxy)phenyl)-7-(1H-pyrazol-4-yl)thieno[3,2-d]py-rimidin-2-amine99. N-(2,4-dimethoxyphenyl)-7-phenylthieno[3,2-d]pyrimidin-2-amine 100.7-bromo-N-(3,4-dimethoxyphenyl)thieno[3,2-d]pyrimidin-2-amine 101.N-(3,4-dimethoxyphenyl)-7-phenylthieno[3,2-d]pyrimidin-2-amine

R348 (Rigel) is defined in Velotta et al., “A novel JAK3 inhibitor,R348, attenuates chronic airway allograft rejection,” Transplantation.2009 Mar. 15; 87(5):653-9.

The present invention also relates to the use of pharmaceuticallyacceptable acid addition salts of compounds of Formulas A and B, as wellas the additional JAK inhibitors described herein. The acids which areused to prepare the pharmaceutically acceptable acid addition salts ofthe aforementioned base compounds of this invention are those which formnon-toxic acid addition salts, i.e., salts containing pharmacologicallyacceptable anions, such as the hydrochloride, hydrobromide, hydroiodide,nitrate, sulfate, bisulfate, phosphate, acid phosphate, acetate,lactate, citrate, acid citrate, tartrate, bitartrate, succinate,maleate, fumarate, gluconate, saccharate, benzoate, methanesulfonate,ethanesulfonate, benzencsulfonate, p-toluenesulfonate and pamoate [i.e.,1,1′-methylene-bis-(2-hydroxy-3-naphthoate)]salts.

The invention also relates to the use of base addition salts of FormulasA and B. The chemical bases that may be used as reagents to preparepharmaceutically acceptable base salts of those compounds of Formulas Aand B that are acidic in nature are those that form non-toxic base saltswith such compounds. Such non-toxic base salts include, but are notlimited to those derived from such pharmacologically acceptable cationssuch as alkali metal cations (e.g., potassium and sodium) and alkalineearth metal cations (e.g., calcium and magnesium), ammonium orwater-soluble amine addition salts such asN-methylglucamine-(meglumine), and the lower alkanolammonium and otherbase salts of pharmaceutically acceptable organic amines.

The JAK inhibitors described herein include all conformational isomers(e.g., cis and trans isomers. Those compounds which have asymmetriccenters exist in different enantiomeric and diastereomeric forms. Thisinvention relates to the use of all optical isomers and stereoisomers ofthe compounds, and mixtures thereof, and to all pharmaceuticalcompositions and methods of treatment that may employ or contain them.In this regard, the invention includes both the E and Z configurations.The compounds of Formulas A and B can also exist as tautomers. Thisinvention relates to the use of all such tautomers and mixtures thereof.

This invention also encompasses pharmaceutical compositions containingprodrugs of compounds of the Formulas A and B. This invention alsoencompasses methods of treating or preventing viral infections that canbe treated or prevented by inhibitors of protein kinases, such as theenzyme Janus Kinase 1, 2, or 3 comprising administering prodrugs ofcompounds of the Formulas A and B. Compounds of Formulas A and B havingfree amino, amido, hydroxy or carboxylic groups can be converted intoprodrugs. Prodrugs include compounds wherein an amino acid residue, or apolypeptide chain of two or more (e.g., two, three or four) amino acidresidues which are covalently joined through peptide bonds to freeamino, hydroxy or carboxylic acid groups of compounds of Formulas A andB. The amino acid residues include the 20 naturally occurring aminoacids commonly designated by three letter symbols and also include,4-hydroxyproline, hydroxylysine, demosine, isodemosine,3-methylhistidine, norvlin, beta-alanine, gamma-aminobutyric acid,citrulline, homocysteine, homoserine, ornithine and methioine sulfone.Prodrugs also include compounds wherein carbonates, carbamates, amidesand alkyl esters which are covalently bonded to the above substituentsof Formulas A and B through the carbonyl carbon prodrug sidechain.

The JAK inhibitors can be used in combination with additionalanti-retroviral agents, including reverse transcriptase inhibitors, suchas nucleoside reverse transcriptase inhibitors (NRTI) and non-nucleosidereverse transcriptase inhibitors (NNRTI), non-nucleoside viralpolymerase inhibitors, protease inhibitors, fusion inhibitors, entryinhibitors, attachment inhibitors, and integrase inhibitors such asraltegravir (Isentress) or MK-0518, GS-9137 (Elvitegravir, GileadSciences), GS-8374 (Gilead Sciences), or GSK-364735.

In one embodiment, the combinations include, in addition to a JAKinhibitor as described herein, at least one adenine nucleoside antiviralagent, at least one cytosine nucleoside antiviral agent, at least oneguanine nucleoside antiviral agent, and at least one thymidinenucleoside antiviral agent. In one aspect of this embodiment, thetherapeutic combinations include, and further include at least oneadditional agent selected from reverse transcriptase inhibitors,especially non-nucleoside viral polymerase inhibitors, proteaseinhibitors, fusion inhibitors, entry inhibitors, attachment inhibitors,and integrase inhibitors such as raltegravir (Isentress) or MK-0518,GS-9137 (elvitegravir, Gilead Sciences), GS-8374 (Gilead Sciences), orGSK-364735.

Certain JAK inhibitors are also inhibitors of CYP3A4, which means thatthey will significantly increase the C_(max) plasma level of anyanti-HIV drug that binds to CYP3A4, including HIV-1 protease inhibitors.

It is believed that this therapy, particularly when administered at anearly stage in the development of HIV-1 infection, has the possibilityof eliminating HIV-1 infection in a patient. While not wishing to bebound to a particular theory, it is believed that the JAK inhibitorsfunction in a way that is not likely to provoke resistance (i.e., doesnot involve inhibition of enzymes, or introduction of modified bases ina way that would provoke enzyme mutations).

Further, when the JAK inhibitors are combined with different nucleosidescontaining all the possible bases (ACTG), optionally in the presence ofadditional agents, the combination minimizes the ability of the virus toadapt its reverse transcriptase and develop resistance to any class ofnucleoside antiviral nucleosides (i.e., adenine, cytosine, thymidine, orguanine), because it would be susceptible to at least one of the othernucleoside antiviral agents that are present, and/or the additionalnon-NRTI therapeutic agent. Furthermore, hitting the same target such asthe active site of the HIV-1 polymerase with different bases allowscomplete and thorough chain termination of all the possible growingviral DNA chains. The use of an NNRTI in addition to the four differentnucleosides (ACTG analogs) can be even more effective, since NNRTI bindto the HIV-polymerase and cause the enzyme to change conformationpreventing chain elongation by natural nucleosides interacting in theactive site of the enzyme.

In any of these embodiments, additional therapeutic agents can be usedin combination with these agents, particularly including agents with adifferent mode of attack. Such agents include but are not limited to:antivirals, such as cytokines, e.g., rIFN alpha, rIFN beta, rIFN gamma;amphotericin B as a lipid-binding molecule with anti-HIV activity; aspecific viral mutagenic agent (e.g., ribavirin), an HIV-1 VIFinhibitor, and an inhibitor of glycoprotein processing. Representativeanti-TNF alpha therapies include, but are not limited to, Infliximab(Remicade), adalimumab (Humira), certolizumab pegol (Cimzia), andgolimumab (Simponi), alone or with a circulating receptor fusion proteinsuch as etanerecpt (Enbrel).

When administered in combination, the agents can be administered in asingle or in multiple dosage forms. In some embodiments, some of theantiviral agents are orally administered, whereas other antiviral agentsare administered by injection, which can occur at around the same time,or at different times.

The compounds can be used in different ways to treat or prevent HIV,and, in one embodiment, to cure an HIV infection. The inventionencompasses combinations of the two types of antiviral agents, orpharmaceutically acceptable derivatives thereof, that are synergistic,i.e., better than either agent or therapy alone.

In one embodiment, a combination of a JAK inhibitor as described herein,a macrophage depleting agent (e.g., clodronate-loaded liposomes,gadolinium chloride (GdCl)), plus HAART therapy is used.

In another embodiment, a combination of a histone deacetylase inhibitor(HDAC inhibitor) or interleukin 7 (IL-7) and HAART and a JAK inhibitoris used.

In another embodiment, the JAK inhibitors are administered to a patientbefore, during, or after administration of a vaccine or animmunomodulatory agent.

Combinations of these approaches can also be used.

The antiviral combinations described herein provide means of treatmentwhich can not only reduce the effective dose of the individual drugsrequired for antiviral activity, thereby reducing toxicity, but can alsoimprove their absolute antiviral effect, as a result of attacking thevirus through multiple mechanisms. That is, various combinationsdescribed herein are useful because their synergistic actions permit theuse of less drug, and/or increase the efficacy of the drugs when usedtogether in the same amount as when used alone.

The use of JAK inhibitors, alone or in combination, provides a means forcircumventing the development of viral resistance, thereby providing theclinician with a more efficacious treatment.

The disclosed JAK inhibitors, used alone or in combination or inalternation therapies, are useful in the prevention and treatment ofHIV-1 infections and other related conditions such as AIDS-relatedcomplex (ARC), persistent generalized lymphadenopathy (PGL),AIDS-related neurological conditions, anti-HIV antibody positive andHIV-positive conditions, Kaposi's sarcoma, thrombocytopenia purpurea andopportunistic infections. In addition, these compounds or formulationscan be used prophylactically to prevent or retard the progression ofclinical illness in individuals who are anti-HIV antibody or HIV-antigenpositive or who have been exposed to HIV. The therapy can be also usedto treat other viral infections, such as HIV-2.

The invention includes methods for treating or preventing, and uses forthe treatment or prophylaxis, of a Flaviviridae infection, including allmembers of the Hepacivirus genus (HCV). Pestivirus genus (BVDV, CSFV,BDV), or Flavivirus genus (Dengue virus, Japanese encephalitis virusgroup (including West Nile Virus), and Yellow Fever virus), as well asAlphaviruscs, such as the Chikungunya virus.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a chart showing the potency and toxicity of JAK inhibitorsTofacitinib or Jakafi versus FDA approved controls AZT and 3TC inacutely infected resting macrophages (MØ), as well as in peripheralblood mononuclear (PBM) cells. Median effective antiviral concentration(EC₅₀) data (potency) is shown in terms of μM concentration of thecompounds. The IC₅₀ values (toxicity) (μM) are also shown in PBM, MØcells, CEM cells, and Vero cells.

FIG. 2 is a chart showing the effect of various concentrations ofTofacitinib and Jakafi on cellular proliferation [total cell number (10⁶cells) versus μM drug] in activated PBM cells incubated for 5 days withthe compounds. Cycloheximide is shown as a positive control, and a“cells plus media” control for each compound is also shown.

FIG. 3 is a chart showing the effect of various concentrations ofTofacitinib and Jakafi on cellular viability (% viability versus μMdrug) in activated PBM cells incubated for 5 days with the compounds.Cycloheximide is shown as a positive control, and a “cells plus media”control for each compound is shown as well.

FIGS. 4a-f show the results of flow cytometric analysis of PHA+IL-2stimulated primary human lymphocytes exposed to various concentrationsof Jakafi or Tofacitinib for 5 days prior to assessment of viabilityusing propidium iodide (flow cytometry). Histograms and scatter plotsare representative data from at least 3 independent experimentsconducted with pooled cells from 8 donors.

FIG. 4a is a scatter plot showing a Side Scatter (SSC) Gating strategy,where the X-axis in the first chart is Side Scatter Pulse Height (SSC-h)and the Y-axis is Side Scatter Pulse Width (SSC-w), and in the secondchart, the forward-scattered light (FSC) is shown with the X axis beingForward Scatter Pulse Height (FSC-H) and the Y axis being ForwardScatter Pulse Width (FSC-W) and Gating strategy based on forward scatter(FSC) and side scatter (SSC) was established and used uniformly acrossall samples (A).

FIG. 4b is a histogram showing the results of flow cytometry studiesusing Propidium Iodide stain, which is read by the phycoerythrin (PE-A)channel, looking at the cell counts of viable cells. Propidium iodide isa large molecule, which exclusively intercalates into the DNA ofdead/dying cells and is detectable by PE fluorescence (flow cytometry).Living cells do not uptake Propidium Iodide, therefore they are notfluorescent or detectable by the PE channel. Cells incubated in theabsence of drug were 92.8% viable (therefore 92.8% of these cells didnot uptake the Propidium Iodide stain), and cells exposed to 95° C. heatfor 1 minute (positive control for dead cells) were 2.8% viable(therefore only 2.8% of cells were negative for Propidium Iodide stain,whereas 97.2% were dead, and therefore positive for Propidium Iodidestain) (B). The data is shown in terms of total percent of cells in eachsample, where gating was established based on viable cells cultured inthe absence of drug.

FIG. 4c shows histograms comparing the cell viability for cells exposedto Jakafi and to no drug (i.e., controls) for concentrations of 0.1 μMJakafi, 1.0 μM Jakafi, 10 μM Jakafi, and 50 μM Jakafi.

FIG. 4d shows histograms comparing the cell viability for cells exposedto Tofacitinib and to no drug (i.e., controls) for concentrations of 0.1μM Tofacitinib, 1.0 μM Tofacitinib, 10 μM Tofacitinib, and 50 μMTofacitinib.

FIGS. 4e and 4f are charts showing the mean and standard deviations fromthe experiments shown in FIGS. 4c (Jakafi) and 4 d (Tofacitinib),respectively.

FIGS. 5a and 5b are charts showing the percent inhibition of HIV-1replication versus untreated control for the co-administration of Jakafiand Tofacitinib in primary human lymphocytes (FIG. 5a ) and macrophages(FIG. 5b ). The data is shown in terms of percent inhibition (%) on theY axis versus drug concentration (μM) on the X axis.

FIGS. 6a and 6b are charts showing the fold increase 50 (FI₅₀) and foldincrease 90 (FI₉₀) for Jakafi and Tofacitinib against variousNRTI-resistant HIV-1 in primary human lymphocytes. Results with NRTIAZT, (−) FTC, 3TC, D4T, ddI, EFV, and TDF are also shown.

FIGS. 7a-7d are charts showing the effect of various Jak inhibitors(Cycloheximide (black line), Tofacitinib (grey line), and Jakafi (dashedline) on proliferation and viability of PHA (FIGS. 7a and 7c ) orPHA+IL-2 (FIGS. 7b and 7d ) stimulated primary human lymphocytes. FIGS.7a and 7b are shown in terms of % viable cells versus concentration ofJak inhibitor (μM). FIGS. 7c and 7d are shown in terms of cell count(10⁶ cells) versus concentration of Jak inhibitor (μM).

FIGS. 8a and 8b are charts showing that Tofacitinib and Jakafi inhibitreactivation of latent HIV-1. FIG. 8a shows the results in a primarycentral memory-based T cell latency model (Bosque and Planelles (2009)Induction of HIV-1 latency and reactivation in primary memory CD4+ Tcells. Blood 113: 58-65), in terms of the % inhibition of reactivationof latent HIV-1 versus concentration of Jak inhibitor (μM). FIG. 8bshows the results in a J-Lat latency T cell system (Jordan et al, (2003)HIV reproducibly establishes a latent infection after acute infection ofT cells in vitro. The EMBO Journal. Vol. 22 No. 8 pp. 1868±1877), interms of the % inhibition of reactivation of latent HIV-1 versusconcentration of Jak inhibitor (μM). Diamonds represent results forTofacitinib, and squares represent results for Jakafi.

FIGS. 9a and 9b are charts showing that Tofacitinib and Jakafi inhibitreactivation of latent HIV-1 in primary human macrophages. Tofacitinib(FIG. 9a ) and Jakafi (FIG. 9b ) inhibit reactivation of latent HIV-1 inprimary human macrophages when drug is applied to cells duringreactivation but removed thereafter. Tofacitinib inhibits ˜40% ofreactivation while Jakafi inhibits ˜35% of reactivation within 72 hrpost reactivation.

FIGS. 10a-10c are charts showing the percent inhibition of PSTAT1,PSTAT3, and PSTAT5, respectively, versus no drug (control) versusmicromolar Jak inhibitor. The lines shown with diamonds represent Jakinhibitor Tofacitinib, and the lines shown with squares represent Jakinhibitor Jakafi.

FIGS. 11 A-E are graphicals showing that Jak inhibitors reduce frequencyof cells harboring integrated viral DNA and IL-15-induced reactivationof latent HIV-1 in CD4 T cells. CD4 T cells were isolated from viremicdonors and incubated with CD3/CD28 plus 0.01, 0.1, 1.0 or 10 μM of Jakinhibitors with or without EC₉₉ of ART (180 nM zidovudine, 100 nMefavirenz, 200 nM Raltegravir) (A and B). After six days, integratedviral DNA was quantified using ultra sensitive Alu PCR versus DMSOcontrols (n=5). 0.01 μM represents the average of all assays completedusing % DMSO equivalent to Jak inhibitor concentrations. Error barsrepresent S.E.M. and statistical significance determined by two-wayANOVA followed by Sidak's multiple comparison post-test: *p<0.05,**p<0.01, ***p<0.001 and ****p<0.0001 for A and B. In panel C-E, memoryCD4+ T cells were isolated from ART treated aviremic donors (n=3),activated with 10 ng/ml IL-15 (panel D) or CD3/CD28 (panel E) andmaintained with or without 1 μM ruxolitinib in the presence of ART. Sixdays post reactivation, extracellular viral RNA copies were quantifiedby qRT-PCR (*p<0.01, one-way ANOVA).

FIG. 12 are graphicals demonstrating that Jak inhibitors block bystanderinfection in primary CD4 T cells. Uninfected CD4⁺ T cells were incubatedwith or without cell trace violet (CTV) dye. Cells with CTV dye werestimulated with CD3/CD28 and various concentrations of ruxolitinib orDMSO for 3 days (A, top). Cells without tracer violet dye were incubatedwith CD3/CD28 for 3 days followed by a 2 hours spinoculation with areplication competent eGFP N14-3 X4 HIV-1 (A, bottom). Afterspinocualtion on Day 3, both cultures (traced and untraced) wereco-incubated for two days in the absence of ruxolitinib. Ruxolitinibinhibits bystander infection (GFP and CTV double positive) of uninfectedbystander cells (CTV⁺) in a dose dependent manner (B and C, n=3).Ruxolitinib blocks proliferation (CTV-lo) of bystander cells in a dosedependent manner with all concentrations tested (B, C). 0.0 μMrepresents the average of all assays completed using % DMSO equivalentto Jak inhibitor concentrations. Error bars represent mean with S.E.M ormean with standard deviation and statistical significance determined bytwo-way ANOVA followed by Sidak's multiple comparison post-test(****p<0.0001) or a two-tailed paired T test (*p<0.005).

FIG. 13: Tabular summary showing that baricitinib blocks the HIVestablishment, maintenance, lifespan, and HIV-induced inflammatoryevents (HIV-associated neurocognitive impairment system) in primaryhuman macrophages and monocytes. Primary human macrophages or monocyteswere infected with HIV-1 BaL and effect of baricitinib on antiviralpotency, HIV-induced inflammation/activation, and reservoir lifespan,maintenance, and expansion was evaluated. Baricitinib blocks HIVreservoir establishment, maintenance, and expansion in primary myeloidcells, and blocks “HAND” inflammatory events that are induced by HIV inkey primary CNS subsets that drive HIV-induced or HIV associatedinflammatory dysfunction in the CNS (macrophages, monocytes).

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to compounds, compositions and methodsfor treating viral infections, such as HIV infections, including HIV-1and HIV-2 infections. In one embodiment, the compounds are heteroarylsubstituted pyrrolo[2,3-b]pyridines and heteroaryl substitutedpyrrolo[2,3-b]pyrimidines that modulate the activity of Janus kinases(JAK inhibitors).

The various embodiments of the invention are described in more detailbelow, and will be better understood with reference to the followingnon-limiting definitions.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of ordinary skillin the art. All patents, applications, published applications and otherpublications referenced herein are incorporated by reference in theirentirety unless stated otherwise. In the event that there are aplurality of definitions for a term herein, those in this sectionprevail unless stated otherwise.

As used herein, any “R” group(s) such as, without limitation, R¹,R^(1a), R^(1b), R^(c) and R^(1d) represent substituents that can beattached to the indicated atom. A non-limiting list of R groups include,but are not limited to, hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl,cycloalkenyl, cycloalkynyl, aryl, heteroaryl, heteroalicyclyl, aralkyl,heteroaralkyl, (heteroalicyclyl)alkyl, hydroxy, protected hydroxy,alkoxy, aryloxy, acyl, ester, mercapto, cyano, halogen, thiocarbonyl,O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido,N-amido, S-sulfonamido, N-sulfonamide, C-carboxy, protected C-carboxy,O-carboxy, isocyanato, thiocyanato, isothiocyanato, nitro, silyl,sulfenyl, sulfinyl, sulfonyl, haloalkyl, haloalkoxy,trihalomethanesulfonyl, trihalomethanesulfonamido, and amino, includingmono- and di-substituted amino groups, and the protected derivativesthereof. An R group may be substituted or unsubstituted. If two “R”groups are covalently bonded to the same atom or to adjacent atoms, thenthey may be “taken together” as defined herein to form a cycloalkyl,cycloalkenyl, cycloalkynyl, aryl, heteroaryl or heteroalicyclyl group.For example, without limitation, if R′ and R″ of an NR′R″ group areindicated to be “taken together”, it means that they are covalentlybonded to one another at their terminal atoms to form a ring thatincludes the nitrogen:

Whenever a group is described as being “optionally substituted” thatgroup may be unsubstituted or substituted with one or more of theindicated substituents. Likewise, when a group is described as being“unsubstituted or substituted” if substituted, the substituent may beselected from one or more the indicated substituents. If no substituentsare indicated, it is meant that the indicated “optionally substituted”or “substituted” group may be substituted with one or more group(s)individually and independently selected from alkyl, alkenyl, alkynyl,cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl,heteroalicyclyl, aralkyl, heteroaralkyl, (heteroalicyclyl)alkyl,hydroxy, protected hydroxyl, alkoxy, aryloxy, acyl, ester, mercapto,alkylthio, arylthio, cyano, halogen, thiocarbonyl, O-carbamyl,N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido, N-amido,S-sulfonamido, N-sulfonamido, C-carboxy, protected C-carboxy, O-carboxy,isocyanato, thiocyanato, isothiocyanato, nitro, silyl, sulfenyl,sulfinyl, sulfonyl, haloalkyl, haloalkoxy, trihalomethanesulfonyl,trihalomethanesulfonamido, and amino, including mono- and di-substitutedamino groups, and the protected derivatives thereof. Each of thesesubstituents can be further substituted.

As used herein, “C to C_(b)” in which “a” and “b” are integers refer tothe number of carbon atoms in an alkyl, alkenyl or alkynyl group, or thenumber of carbon atoms in the ring of a cycloalkyl, cycloalkenyl,cycloalkynyl, aryl, heteroaryl or heteroalicyclyl group. That is, thealkyl, alkenyl, alkynyl, ring of the cycloalkyl, ring of thecycloalkenyl, ring of the cycloalkynyl, ring of the aryl, ring of theheteroaryl or ring of the heteroalicyclyl can contain from “a” to “b”,inclusive, carbon atoms. Thus, for example, a “C₁ to C₄ alkyl” grouprefers to all alkyl groups having from 1 to 4 carbons, that is, CH₃—,CH₃CH₂—, CH₃CH₂CH₂—, (CH₃)₂CH—, CH₃CH₂CH₂CH₂—, CH₃CH₂CH(CH₃)— and(CH₃)₃C—. If no “a” and “b” are designated with regard to an alkyl,alkenyl, alkynyl, cycloalkyl cycloalkenyl, cycloalkynyl, aryl,heteroaryl or heteroalicyclyl group, the broadest range described inthese definitions is to be assumed.

As used herein, the term “alkyl” can be straight or branched hydrocarbonchains that comprise a fully saturated (no double or triple bonds)hydrocarbon group. The alkyl group may have 1 to 20 carbon atoms(whenever it appears herein, a numerical range such as “1 to 20” refersto each integer in the given range; e.g., “1 to 20 carbon atoms” meansthat the alkyl group may consist of 1 carbon atom, 2 carbon atoms, 3carbon atoms, etc., up to and including 20 carbon atoms, although thepresent definition also covers the occurrence of the term “alkyl” whereno numerical range is designated). The alkyl group may also be a mediumsize alkyl having 1 to 10 carbon atoms. The alkyl group can also be alower alkyl having 1 to 6 carbon atoms. The alkyl group of the compoundsmay be designated as “C₁-C₆ alkyl” or similar designations. By way ofexample only, “C₁-C₄ alkyl” indicates that there are one to four carbonatoms in the alkyl chain, i.e., the alkyl chain is selected from methyl,ethyl, propyl, iso-propyl, n-butyl, iso-butyl, sec-butyl, and t-butyl.By way of example only, “C₁-C₆ alkyl” indicates that there are one tosix carbon atoms in the alkyl chain. Typical alkyl groups include, butare in no way limited to, methyl, ethyl, propyl, isopropyl, butyl,isobutyl, tertiary butyl, pentyl, hexyl, and the like. The alkyl groupmay be substituted or unsubstituted.

As used herein, “alkenyl” refers to an alkyl group that contains in thestraight or branched hydrocarbon chain one or more double bonds. Analkenyl group may be unsubstituted or substituted.

As used herein, “alkynyl” refers to an alkyl group that contains in thestraight or branched hydrocarbon chain one or more triple bonds. Analkynyl group may be unsubstituted or substituted.

As used herein, the term “alkoxy” includes O-alkyl groups wherein“alkyl” is defined above. As used herein, “cycloalkyl” refers to acompletely saturated (no double or triple bonds) mono- or multi-cyclichydrocarbon ring system. When composed of two or more rings, the ringsmay be joined together in a fused fashion. Cycloalkyl groups can contain3 to 10 atoms in the ring(s) or 3 to 8 atoms in the ring(s). Acycloalkyl group may be unsubstituted or substituted. Typical cycloalkylgroups include, but are in no way limited to, cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, and the like.

As used herein, “cycloalkenyl” refers to a mono- or multi-cyclichydrocarbon ring system that contains one or more double bonds in atleast one ring; although, if there is more than one, the double bondscannot form a fully delocalized pi-electron system throughout all therings (otherwise the group would be “aryl,” as defined herein). Whencomposed of two or more rings, the rings may be connected together in afused fashion. A cycloalkenyl group may be unsubstituted or substituted.

As used herein, “cycloalkynyl” refers to a mono- or multi-cyclichydrocarbon ring system that contains one or more triple bonds in atleast one ring. Pf there is more than one triple bond, the triple bondscannot form a fully delocalized pi-electron system throughout all therings. When composed of two or more rings, the rings may be joinedtogether in a fused fashion. A cycloalkynyl group may be unsubstitutedor substituted.

As used herein, “aryl” refers to a carbocyclic (all carbon) monocyclicor multicyclic aromatic ring system (including fused ring systems wheretwo carbocyclic rings share a chemical bond) that has a fullydelocalized pi-electron system throughout all the rings. The number ofcarbon atoms in an aryl group can vary. For example, the aryl group is aC₆₋₁₄ aryl group, a C₆₋₁₀ aryl group, or a C₆ aryl group. Examples ofaryl groups include, but are not limited to, benzene, naphthalene andazulene. An aryl group may be substituted or unsubstituted.

As used herein, “heteroaryl” refers to a monocyclic or multicyclicaromatic ring system (a ring system with fully delocalized pi-electronsystem) that contain(s) one or more heteroatoms, that is, an elementother than carbon, including but not limited to, nitrogen, oxygen andsulfur. The number of atoms in the ring(s) of a heteroaryl group canvary. For example, the heteroaryl group can contain 4 to 14 atoms in thering(s), 5 to 10 atoms in the ring(s) or 5 to 6 atoms in the ring(s).Furthermore, the term “heteroaryl” includes fused ring systems where tworings, such as at least one aryl ring and at least one heteroaryl ring,or at least two heteroaryl rings, share at least one chemical bond.Examples of heteroaryl rings include, but are not limited to, furan,furazan, thiophene, benzothiophene, phthalazine, pyrrole, oxazole,benzoxazole, 1,2,3-oxadiazole, 1,2,4-oxadiazole, thiazole,1,2,3-thiadiazole, 1,2,4-thiadiazole, benzothiazole, imidazole,benzimidazole, indole, indazole, pyrazole, benzopyrazole, isoxazole,benzoisoxazole, isothiazole, triazole, benzotriazole, thiadiazole,tetrazole, pyridine, pyridazine, pyrimidine, pyrazine, purine,pteridine, quinoline, isoquinoline, quinazoline, quinoxaline, cinnoline,and triazine. A heteroaryl group may be substituted or unsubstituted.

As used herein, “heteroalicyclic” or “heteroalicyclyl” refers to three-,four-, five-, six-, seven-, eight-, nine-, ten-, up to 18-memberedmonocyclic, bicyclic, and tricyclic ring system wherein carbon atomstogether with from 1 to 5 heteroatoms constitute said ring system. Aheterocycle may optionally contain one or more unsaturated bondssituated in such a way, however, that a fully delocalized pi-electronsystem does not occur throughout all the rings. The heteroatoms areindependently selected from oxygen, sulfur, and nitrogen. A heterocyclemay further contain one or more carbonyl or thiocarbonylfunctionalities, so as to make the definition include oxo-systems andthio-systems such as lactams, lactones, cyclic imides, cyclicthioimides, cyclic carbamates, and the like. When composed of two ormore rings, the rings may be joined together in a fused fashion.Additionally, any nitrogens in a heteroalicyclic may be quaternized.Heteroalicyclyl or heteroalicyclic groups may be unsubstituted orsubstituted. Examples of such “heteroalicyclic” or “heteroalicyclyl”groups include but are not limited to, 1,3-dioxin, 1,3-dioxane,1,4-dioxane, 1,2-dioxolane, 1,3-dioxolane, 1,4-dioxolane, 1,3-oxathiane,1,4-oxathiin, 1,3-oxathiolane, 1,3-dithiole, 1,3-dithiolane,1,4-oxathiane, tetrahydro-1,4-thiazine, 2H-1,2-oxazine, maleimide,succinimide, barbituric acid, thiobarbituric acid, dioxopiperazine,hydantoin, dihydrouracil, trioxane, hexahydro-1,3,5-triazine,imidazoline, imidazolidine, isoxazoline, isoxazolidine, oxazoline,oxazolidine, oxazolidinone, thiazoline, thiazolidine, morpholine,oxirane, piperidine N-Oxide, piperidine, piperazine, pyrrolidine,pyrrolidone, pyrrolidione, A-piperidone, pyrazoline, pyrazolidine,2-oxopyrrolidine, tetrahydropyran, 4H-pyran, tetrahydrothiopyran,thiamorpholine, thiamorpholine sulfoxide, thiamorpholine sulfone, andtheir benzo-fused analogs (e.g., benzimidazolidinone,tetrahydroquinoline, 3,4-methylenedioxyphenyl).

An “aralkyl” is an aryl group connected, as a substituent, via a loweralkylene group. The lower alkylene and aryl group of an aralkyl may besubstituted or unsubstituted. Examples include but are not limited tobenzyl, substituted benzyl, 2-phenylalkyl, 3-phenylalkyl, andnaphtylalkyl.

A “heteroaralkyl” is heteroaryl group connected, as a substituent, via alower alkylene group. The lower alkylene and heteroaryl group ofheteroaralkyl may be substituted or unsubstituted. Examples include butare not limited to 2-thienylalkyl, 3-thienylalkyl, furylalkyl,thienylalkyl, pyrrolylalkyl, pyridylalkyl, isoxazolylalkyl, andimidazolylalkyl, and their substituted as well as benzo-fused analogs.

A “(heteroalicyclyl)alkyl” is a heterocyclic or a heteroalicyclylicgroup connected, as a substituent, via a lower alkylene group. The loweralkylene and heterocyclic or a heterocyclyl of a (heteroalicyclyl)alkylmay be substituted or unsubstituted. Examples include but are notlimited tetrahydro-2H-pyran-4-yl)methyl, (piperidin-4-yl)ethyl,(piperidin-4-yl)propyl, (tetrahydro-2H-thiopyran-4-yl)methyl, and(1,3-thiazinan-4-yl)methyl.

“Lower alkylene groups” are straight-chained tethering groups, formingbonds to connect molecular fragments via their terminal carbon atoms.Examples include but are not limited to methylene (—CH₂—), ethylene(—CH₂CH₂—), propylene (—CH₂CH₂CH₂—), and butylene (—CH₂CH₂CH₂CH₂—). Alower alkylene group may be substituted or unsubstituted.

As used herein, “alkoxy” refers to the formula —OR wherein R is analkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl,heteroaryl, heteroalicyclyl, aralkyl, or (heteroalicyclyl)alkyl isdefined as above. Examples of include methoxy, ethoxy, n-propoxy,1-methylethoxy (isopropoxy), n-butoxy, iso-butoxy, sec-butoxy,tert-butoxy, phenoxy and the like. An alkoxy may be substituted orunsubstituted.

As used herein, “acyl” refers to a hydrogen, alkyl, alkenyl, alkynyl, oraryl connected, as substituents, via a carbonyl group. Examples includeformyl, acetyl, propanoyl, benzoyl, and acryl. An acyl may besubstituted or unsubstituted.

As used herein, “hydroxyalkyl” refers to an alkyl group in which one ormore of the hydrogen atoms are replaced by hydroxy group. Examples ofhydroxyalkyl groups include but are not limited to, 2-hydroxyethyl,3-hydroxypropyl, 2-hydroxypropyl, and 2,2-dihydroxyethyl. A hydroxyalkylmay be substituted or unsubstituted.

As used herein, “haloalkyl” refers to an alkyl group in which one ormore of the hydrogen atoms are replaced by halogen (e.g.,mono-haloalkyl, di-haloalkyl and tri-haloalkyl). Such groups include butare not limited to, chloromethyl, fluoromethyl, difluoromethyl,trifluoromethyl and 1-chloro-2-fluoromethyl, 2-fluoroisobutyl. Ahaloalkyl may be substituted or unsubstituted.

As used herein, “haloalkoxy” refers to an alkoxy group in which one ormore of the hydrogen atoms are replaced by halogen (e.g.,mono-haloalkoxy, di-haloalkoxy and tri-haloalkoxy). Such groups includebut are not limited to, chloromethoxy, fluoromethoxy, difluoromethoxy,trifluoromethoxy and 1-chloro-2-fluoromethoxy, 2-fluoroisobutoxy. Ahaloalkoxy may be substituted or unsubstituted.

A “sulfenyl” group refers to an “—SR” group in which R is hydrogen,alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl,heteroaryl, heteroalicyclyl, aralkyl, or (heteroalicyclyl)alkyl. Asulfenyl may be substituted or unsubstituted.

A “sulfinyl” group refers to an “—S(═O)—R” group in which R is the sameas defined with respect to sulfenyl. A sulfinyl may be substituted orunsubstituted.

A “sulfonyl” group refers to an “SO₂R” group in which R is the same asdefined with respect to sulfenyl. A sulfonyl may be substituted orunsubstituted.

An “O-carboxy” group refers to a “RC(═O)O—” group in which R ishydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl,cycloalkynyl, aryl, heteroaryl, heteroalicyclyl, aralkyl, or(heteroalicyclyl)alkyl, as defined herein. An O-carboxy may besubstituted or unsubstituted.

The terms “ester” and “C-carboxy” refer to a “—C(═O)OR” group in which Ris the same as defined with respect to O-carboxy. An ester and C-carboxymay be substituted or unsubstituted.

A “thiocarbonyl” group refers to a “—C(═S)R” group in which R is thesame as defined with respect to O-carboxy. A thiocarbonyl may besubstituted or unsubstituted.

A “trihalomethanesulfonyl” group refers to an “X₃CSO₂—” group wherein Xis a halogen.

A “trihalomethanesulfonamido” group refers to an “X₃CS(O)₂RN—” groupwherein X is a halogen and R defined with respect to O-carboxy.

The term “amino” as used herein refers to a —NH₂ group.

As used herein, the term “hydroxy” refers to a —OH group.

A “cyano” group refers to a “—CN” group.

The term “azido” as used herein refers to a —N₃ group.

An “isocyanato” group refers to a “—NCO” group.

A “thiocyanato” group refers to a “—CNS” group.

An “isothiocyanato” group refers to an “—NCS” group.

A “mercapto” group refers to an “—SH” group.

A “carbonyl” group refers to a C═O group.

An “S-sulfonamido” group refers to a “—SO₁NR_(A)R_(B)” group in whichR_(A) and R_(B) are the same as R defined with respect to O-carboxy. AnS-sulfonamido may be substituted or unsubstituted.

An “N-sulfonamido” group refers to a “R_(B)SO₂N(R_(A))—” group in whichR_(A) and R_(B) are the same as R defined with respect to O-carboxy. AN-sulfonamido may be substituted or unsubstituted.

An “O-carbamyl” group refers to a “—OC(═O)NR_(A)R_(B)” group in whichR_(A) and R_(B) are the same as R defined with respect to O-carboxy. AnO-carbamyl may be substituted or unsubstituted.

An “N-carbamyl” group refers to an “R_(B)OC(═O)NR_(A)—” group in whichR_(A) and R_(B) are the same as R defined with respect to O-carboxy. AnN-carbamyl may be substituted or unsubstituted.

An “O-thiocarbamyl” group refers to a “—OC(═S)—NR_(A)R_(B)” group inwhich R_(A) and R_(B) are the same as R defined with respect toO-carboxy. An O-thiocarbamyl may be substituted or unsubstituted.

An “N-thiocarbamyl” group refers to an “R_(B)OC(═S)NR_(A)—” group inwhich R_(A) and R_(B) are the same as R defined with respect toO-carboxy. An N-thiocarbamyl may be substituted or unsubstituted.

A “C-amido” group refers to a “—C(═O)NR_(A)R_(B)” group in which R_(A)and R_(B) are the same as R defined with respect to O-carboxy. A C-amidocan be substituted or unsubstituted.

An “N-amido” group refers to a “R_(B)C(═O)NR_(A)—” group in which R_(A)and R_(B) are the same as R defined with respect to O-carboxy. AnN-amido can be substituted or unsubstituted.

As used herein, “organylcarbonyl” refers to a group of the formula—C(═O)R′ wherein R′ can be alkyl, alkenyl, alkynyl, cycloalkyl,cycloalkenyl, cycloalkynyl, aryl, heteroaryl, heteroalicyclyl, aralkyl,or (heteroalicyclyl)alkyl. An organylcarbonyl can be substituted orunsubstituted.

The term “alkoxycarbonyl” as used herein refers to a group of theformula —C(═O)OR′, wherein R′ is the same as defined with respect toorganylcarbonyl. An alkoxycarbonyl can be substituted or unsubstituted.

As used herein, “organylaminocarbonyl” refers to a group of the formulaC(═O)NR′R″ wherein R′ and R″ are independently selected from the samesubstituents as defined with respect to organylcarbonyl. Anorganylaminocarbonyl can be substituted or unsubstituted.

As used herein, the term “levulinoyl” refers to a —C(═O)CH₂CH₂C(═O)CH₃group.

The term “halogen atom,” as used herein, means any one of theradio-stable atoms of column 7 of the Periodic Table of the Elements,i.e., fluorine, chlorine, bromine, or iodine, with fluorine and chlorinebeing preferred.

Where the numbers of substituents is not specified (e.g. haloalkyl),there may be one or more substituents present. For example “haloalkyl”may include one or more of the same or different halogens. As anotherexample, “C₁-C₃ alkoxyphenyl” may include one or more of the same ordifferent alkoxy groups containing one, two or three atoms.

As used herein, the term “nucleoside” refers to a compound composed ofany pentose or modified pentose moiety attached to a specific portion ofa heterocyclic base, tautomer, or derivative thereof such as the9-position of a purine, 1-position of a pyrimidine, or an equivalentposition of a heterocyclic base derivative. Examples include, but arenot limited to, a ribonucleoside comprising a ribose moiety and adeoxyribonucleoside comprising a deoxyribose moiety, and in someinstances, the nucleoside is a nucleoside drug analog. As used herein,the term “nucleoside drug analog” refers to a compound composed of anucleoside that has therapeutic activity, such as antiviral,antineoplastic, anti-parasitic and/or antibacterial activity.

As used herein, the term “nucleotide” refers to a nucleoside having aphosphate ester substituted on the 5′-position or an equivalent positionof a nucleoside derivative.

As used herein, the term “heterocyclic base” refers to a purine, apyrimidine and derivatives thereof. The term “purine” refers to asubstituted purine, its tautomers and analogs thereof. Similarly, theterm “pyrimidine” refers to a substituted pyrimidine, its tautomers andanalogs thereof. Examples of purines include, but are not limited to,purine, adenine, guanine, hypoxanthine, xanthine, theobromine, caffeine,uric acid and isoguanine. Examples of pyrimidines include, but are notlimited to, cytosine, thymine, uracil, and derivatives thereof. Anexample of an analog of a purine is 1,2,4-triazole-3-carboxamide.

Other non-limiting examples of heterocyclic bases include diaminopurine,8-oxo-N⁶-methyladenine, 7-deazaxanthine, 7-deazaguanine,N⁴,N⁴-ethanocytosin, N⁶,N⁶-ethano-2,6-diaminopurine, 5-methylcytosine,5-fluorouracil, 5-bromouracil, pseudoisocytosine, isocytosine,isoguanine, and other heterocyclic bases described in U.S. Pat. Nos.5,432,272 and 7,125,855, which are incorporated herein by reference forthe limited purpose of disclosing additional heterocyclic bases.

The term “—O-linked amino acid” refers to an amino acid that is attachedto the indicated moiety via its main-chain carboxyl function group. Whenthe amino acid is attached, the hydrogen that is part of the —OH portionof the carboxyl function group is not present and the amino acid isattached via the remaining oxygen. An —O-linked amino acid can beprotected at any nitrogen group that is present on the amino acid. Forexample, an —O-linked amino acid can contain an amide or a carbamategroup. Suitable amino acid protecting groups include, but are notlimited to, carbobenzyloxy (Cbz), p-methoxybenzyl carbonyl (Moz orMeOZ), tert-butyloxycarbonyl (BOC), 9-fluorenylmethyloxycarbonyl (FMOC),benzyl (Bn), p-methoxybenzyl (PMB), 3,4-dimethoxybenzyl (DMPM), andtosyl (Ts) groups. The term “—N-linked amino acid” refers to an aminoacid that is attached to the indicated moiety via its main-chain aminoor mono-substituted amino group. When the amino acid is attached in an—N-linked amino acid, one of the hydrogens that is part of themain-chain amino or mono-substituted amino group is not present and theamino acid is attached via the nitrogen. An —N-linked amino acid can beprotected at any hydroxyl or carboxyl group that is present on the aminoacid. For example, an —N-linked amino acid can contain an ester or anether group. Suitable amino acid protecting groups include, but are notlimited to, methyl esters, ethyl esters, propyl esters, benzyl esters,tert-butyl esters, silyl esters, orthoesters, and oxazoline. As usedherein, the term “amino acid” refers to any amino acid (both standardand non-standard amino acids), including, but limited to, α-amino acidsβ-amino acids, γ-amino acids and δ-amino acids. Examples of suitableamino acids, include, but are not limited to, alanine, asparagine,aspartate, cysteine, glutamate, glutamine, glycine, proline, serine,tyrosine, arginine, histidine, isoleucine, leucine, lysine, methionine,phenylalanine, threonine, tryptophan and valine.

The terms “derivative,” “variant,” or other similar terms refer to acompound that is an analog of the other compound.

The terms “protecting group” and “protecting groups” as used hereinrefer to any atom or group of atoms that is added to a molecule in orderto prevent existing groups in the molecule from undergoing unwantedchemical reactions. Examples of protecting group moieties are describedin T. W Greene and P. G. M. Wuts, Protective Groups in OrganicSynthesis, 3. Ed. John Wiley & Sons (1999), and in J. F. W. McOmie,Protective Groups in Organic Chemistry Plenum Press (1973), both ofwhich are hereby incorporated by reference for the limited purpose ofdisclosing suitable protecting groups The protecting group moiety may bechosen in such a way, that they are stable to certain reactionconditions and readily removed at a convenient stage using methodologyknown from the art. A non-limiting list of protecting groups includebenzyl, substituted benzyl; alkylcarbonyls (e g., t-butoxycarbonyl(BOC)); arylalkylcarbonyls (e.g., benzyloxycarbonyl, benzoyl),substituted methyl ether (e.g. methoxymethyl ether); substituted ethylether, a substituted benzyl ether; tetrahydropyranyl ether; silyl ethers(e.g, tπmethylsilyl, tnethylsilyl, tnisopropylsilyl,t-butyldimethylsilyl, or t-butyldiphenylsilyl), esters (e.g. benzoateester), carbonates (e g. methoxymethylcarbonate), sulfonates (e gtosylate, mesylate), acyclic ketal (e g dimethyl acetal); cyclic ketals(e.g., 1,3-dioxane or 1,3-dioxolanes); acyclic acetal; cyclic acetal,acyclic hemiacetal, cyclic hemiacetal, and cyclic dithioketals (e.g.,1,3-dithiane or 1,3-dithiolane).

“Leaving group” as used herein refers to any atom or moiety that iscapable of being displaced by another atom or moiety in a chemicalreaction. More specifically, in some embodiments, “leaving group” refersto the atom or moiety that is displaced in a nucleophilic substitutionreaction hi some embodiments, “leaving groups” are any atoms or moietiesthat are conjugate bases of strong acids Examples of suitable leavinggroups include, but are not limited to, tosylates and halogensNon-limiting characteristics and examples of leaving groups can befound, for example in Organic Chemistry, 2d ed, Francis Carey (1992),pages 328-331, Introduction to Organic Chemistry, 2d ed., AndrewStreitwieser and Clayton Heathcock (1981), pages 169-171; and OrganicChemistry, 5^(th) ed., John McMurry (2000), pages 398 and 408; all ofwhich are incorporated herein by reference for the limited purpose ofdisclosing characteristics and examples of leaving groups.

As used herein, the abbreviations for any protective groups, ammo acidsand other compounds, are, unless indicated otherwise, in accord withtheir common usage, recognized abbreviations, or the IUPAC-IUBCommission on Biochemical Nomenclature (See, Biochem. 1972 11:942-944).

A “prodrug” refers to an agent that is converted into the parent drug invivo. Prodrugs are often useful because, in some situations, they may beeasier to administer than the parent drug. They may, for instance, bebioavailable by oral administration whereas the parent is not. Theprodrug may also have improved solubility in pharmaceutical compositionsover the parent drug. Examples of prodrugs include compounds that haveone or more biologically labile groups attached to the parent drug(e.g., a compound of Formula 1 and/or a compound of Formula 11). Forexample, one or more biologically labile groups can be attached to afunctional group of the parent drug (for example, by attaching one ormore biologically labile groups to a phosphate). When more than onebiologically labile groups is attached, the biologically labile groupscan be the same or different. The biologically labile group(s) can belinked (for example, through a covalent bond), to an oxygen or aheteroatom, such as a phosphorus of a monophosphate, diphosphate,triphosphate, and/or a stabilized phosphate analog containing carbon,nitrogen or sulfur (referred to hereinafter in the present paragraph as“phosphate”). In instances where the prodrug is form by attaching one ormore biologically labile groups to the phosphate, removal of thebiologically labile group in the host produces a phosphate. The removalof the biologically labile group(s) that forms the prodrug can beaccomplished by a variety of methods, including, but not limited to,oxidation, reduction, amination, deamination, hydroxylation,dehydroxylation, hydrolysis, dehydrolysis, alkylation, dealkylation,acylation, deacylation, phosphorylation, dephosphorylation, hydrationand/or dehydration. An example, without limitation, of a prodrug wouldbe a compound which is administered as an ester (the “prodrug”) tofacilitate transmittal across a cell membrane where water solubility isdetrimental to mobility but which then is metabolically hydrolyzed tothe carboxylic acid, the active entity, once inside the cell wherewater-solubility is beneficial. A further example of a prodrug mightcomprise a short peptide (polyaminoacid) bonded to an acid group wherethe peptide is metabolized or cleaved to reveal the active moiety.Additional examples of prodrug moieties include the following:R*,R*C(═O)OCH₂—, R*C(═O)SCH₂CH₂—, R*C(═O)SCHR′NH—, phenyl-O—, N-linkedamino acids, O-linked amino acids, peptides, carbohydrates, and lipids,wherein each R is independently selected from an alkyl, an alkenyl, analkynyl, an aryl, an aralkyl, acyl, sulfonate ester, a lipid, an—N-linked amino acid, an —O-linked amino acid, a peptide and acholesterol. The prodrug can be a carbonate. The carbonate can be acyclic carbonate. The cyclic carbonate can contain a carbonyl groupbetween two hydroxyl groups that results in the formation of a five orsix membered ring. Conventional procedures for the selection andpreparation of suitable prodrug derivatives are described, for example,in Design of Prodrugs, (ed. H. Bundgaard, Elsevier, 1985), which ishereby incorporated herein by reference for the limited purpose ofdescribing procedures and preparation of suitable prodrug derivatives.

The term “pro-drug ester” refers to derivatives of the compoundsdisclosed herein formed by the addition of any of several ester-forminggroups that are hydrolyzed under physiological conditions. Examples ofpro-drug ester groups include pivaloyloxymethyl, acetoxymethyl,phthalidyl, indanyl and methoxymethyl, as well as other such groupsknown in the art, including a (5-R-2-oxo-1,3-dioxolen-4-yl)methyl group.Other examples of pro-drug ester groups can be found in, for example, T.Higuchi and V. Stella, in “Pro-drugs as Novel Delivery Systems”, Vol.14, A.C.S. Symposium Series, American Chemical Society (1975); and“Bioreversible Carriers in Drug Design: Theory and Application”, editedby E. B. Roche, Pergamon Press: New York, 14-21 (1987) (providingexamples of esters useful as prodrugs for compounds containing carboxylgroups). Each of the above-mentioned references is herein incorporatedby reference for the limited purpose of disclosing ester-forming groupsthat can form prodrug esters.

The term “pharmaceutically acceptable salt” refers to a salt of acompound that does not cause significant irritation to an organism towhich it is administered and does not abrogate the biological activityand properties of the compound. In some embodiments, the salt is an acidaddition salt of the compound. Pharmaceutical salts can be obtained byreacting a compound with inorganic acids such as hydrohalic acid (e.g.,hydrochloric acid or hydrobromic acid), sulfuric acid, nitric acid,phosphoric acid and the like. Pharmaceutical salts can also be obtainedby reacting a compound with an organic acid such as aliphatic oraromatic carboxylic or sulfonic acids, for example acetic, succinic,lactic, malic, tartaric, citric, ascorbic, nicotinic, methanesulfonic,ethanesulfonic, p-toluenesulfonic, salicylic or naphthalenesulfonicacid. Pharmaceutical salts can also be obtained by reacting a compoundwith a base to form a salt such as an ammonium salt, an alkali metalsalt, such as a sodium or a potassium salt, an alkaline earth metalsalt, such as a calcium or a magnesium salt, a salt of organic basessuch as dicyclohexylamine, N-methyl-D-glucamine,tris(hydroxymethyl)methylamine, C₁-C₇ alkylamine, cyclohexylamine,triethanolamine, ethylenediamine, and salts with amino acids such asarginine, lysine, and the like.

The term “protected” as used herein and unless otherwise defined refersto a group that is added to an oxygen, nitrogen, or phosphorus atom toprevent its further reaction or for other purposes. A wide variety ofoxygen and nitrogen protecting groups are known to those skilled in theart of organic synthesis. The term aryl, as used herein, and unlessotherwise specified, refers to phenyl, biphenyl, or naphthyl, andpreferably phenyl. The aryl group can be optionally substituted with oneor more moieties selected from the group consisting of hydroxyl, amino,alkylamino, arylamino, alkoxy, aryloxy, nitro, cyano, sulfonic acid,sulfate, phosphonic acid, phosphate, or phosphonate, either unprotected,or protected as necessary, as known to those skilled in the art, forexample, as taught in Greene, et al., Protective Groups in OrganicSynthesis, John Wiley and Sons, Second Edition, 1991.

The term purine or pyrimidine base includes, but is not limited to,adenine, N⁶-alkylpurines, N⁶-acylpurines (wherein acyl is C(O)(alkyl,aryl, alkylaryl, or arylalkyl), N⁶-benzylpurine, N⁶-halopurine,N⁶-vinylpurine, N⁶-acetylenic purine, N⁶-acyl purine, N⁶-hydroxyalkylpurine, N⁶-thioalkyl purine, N²-alkylpurines, N²-alkyl-6-thiopurines,thymine, cytosine, 5-fluorocytosine, 5-methylcytosine, 6-azapyrimidine,including 6-azacytosine, 2- and/or 4-mercaptopyrmidine, uracil,5-halouracil, including 5-fluorouracil, C⁵-alkylpyrimidines,C⁵-benzylpyrimidines, C⁵-halopyrimidines, C⁵-vinylpyrimidine,C⁵-acetylenic pyrimidine, C⁵-acyl pyrimidine, C⁵-hydroxyalkyl purine,C⁵-amidopyrimidine, C⁵-cyanopyrimidine, C⁵-nitropyrimidine,C⁵-aminopyrimidine, N²-alkylpurines, N²-alkyl-6-thiopurines,5-azacytidinyl, 5-azauracilyl, triazolopyridinyl, imidazolopyridinyl,pyrrolopyrimidinyl, and pyrazolopyrimidinyl. Purine bases include, butare not limited to, guanine, adenine, hypoxanthine, 2,6-diaminopurine,2-chloro-2-aminopurine, inosine, and 6-chloropurine. Functional oxygenand nitrogen groups on the base can be protected as necessary ordesired. Suitable protecting groups are well known to those skilled inthe art, and include trimethylsilyl, dimethylhexylsilyl,t-butyldimethylsilyl, and t-butyldiphenylsilyl, trityl, alkyl groups,acyl groups such as acetyl and propionyl, methanesulfonyl, andp-toluenesulfonyl.

The term acyl refers to a carboxylic acid ester in which thenon-carbonyl moiety of the ester group is selected from straight,branched, or cyclic alkyl or lower alkyl, alkoxyalkyl includingmethoxymethyl, aralkyl including benzyl, aryloxyalkyl such asphenoxymethyl, aryl including phenyl optionally substituted withhalogen, C to C₄ alkyl or C₁ to C₁₋₄ alkoxy, sulfonate esters such asalkyl or aralkyl sulphonyl including methanesulfonyl, the mono, di ortriphosphate ester, trityl or monomethoxytrityl, substituted benzyl,trialkylsilyl (e.g. dimethyl-t-butylsilyl) or diphenylmethylsilyl. Arylgroups in the esters optimally comprise a phenyl group. Acyl can alsoinclude a natural or synthetic amino acid moiety.

As used herein, the term “substantially free of” or “substantially inthe absence of” refers to a nucleoside composition that includes atleast 95% to 98%, or more preferably, 99% to 100%, of the designatedenantiomer of that nucleoside.

Similarly, the term “isolated” refers to a nucleoside composition thatincludes at least 85 or 90% by weight, preferably 95% to 98% by weight,and even more preferably 99% to 100% by weight, of the nucleoside, theremainder comprising other chemical species or enantiomers.

The term “host,” as used herein, refers to a unicellular ormulticellular organism in which the virus can replicate, including celllines and animals, and preferably a human. Alternatively, the host canbe carrying a part of the viral genome, whose replication or functioncan be altered by the compounds of the present invention. The term hostspecifically refers to infected cells, cells transfected with all orpart of the viral genome and animals, in particular, primates (includingchimpanzees) and humans. Relative to abnormal cellular proliferation,the term “host” refers to unicellular or multicellular organism in whichabnormal cellular proliferation can be mimicked. The term hostspecifically refers to cells that abnormally proliferate, either fromnatural or unnatural causes (for example, from genetic mutation orgenetic engineering, respectively), and animals, in particular, primates(including chimpanzees) and humans. In most animal applications of thepresent invention, the host is a human patient. Veterinary applications,in certain indications, however, are clearly anticipated by the presentinvention (such as bovine viral diarrhea virus in cattle, hog choleravirus in pigs, and border disease virus in sheep).

The term “halo”, as used herein, unless otherwise indicated, includesfluoro, chloro, bromo or iodo.

The compounds of this invention may contain double bonds. When suchbonds are present, the compounds of the invention exist as cis and transconfigurations and as mixtures thereof.

Unless otherwise indicated, the alkyl and alkenyl groups referred toherein, as well as the alkyl moieties of other groups referred to herein(e.g., alkoxy), may be linear or branched, and they may also be cyclic(e.g., cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl or cycloheptyl)or be linear or branched and contain cyclic moieties. Unless otherwiseindicated, halogen includes fluorine, chlorine, bromine, and iodine.

(C₂-C₉)Heterocycloalkyl when used herein refers to pyrrolidinyl,tetrahydrofuranyl, dihydrofuranyl, tetrahydropyranyl, pyranyl,thiopyranyl, aziridinyl, oxiranyl, methylenedioxyl, chromenyl,isoxazolidinyl, 1,3-oxazolidin-3-yl, isothiazolidinyl,1,3-thiazolidin-3-yl, 1,2-pyrazolidin-2-yl, 1,3-pyrazolidin-1-yl,piperidinyl, thiomorpholinyl, 1,2-tetrahydrothiazin-2-yl,1,3-tetrahydrothiazin-3-yl, tetrahydrothiadiazinyl, morpholinyl,1,2-tetrahydrodiazin-2-yl, 1,3-tetrahydrodiazin-1-yl,tetrahydroazepinyl, piperazinyl, chromanyl, etc. One of ordinary skillin the art will understand that the connection of said(C₂-C₉)heterocycloalkyl rings is through a carbon or a sp³ hybridizednitrogen heteroatom.

(C₂-C₉)Heteroaryl when used herein refers to furyl, thienyl, thiazolyl,pyrazolyl, isothiazolyl, oxazolyl, isoxazolyl, pyrrolyl, triazolyl,tetrazolyl, imidazolyl, 1,3,5-oxadiazolyl, 1,2,4-oxadiazolyl,1,2,3-oxadiazolyl, 1,3,5-thiadiazolyl, 1,2,3-thiadiazolyl,1,2,4-thiadiazolyl, pyridyl, pyrimidyl, pyrazinyl, pyridazinyl,1,2,4-triazinyl, 1,2,3-triazinyl, 1,3,5-triazinyl,pyrazolo[3,4-b]pyridinyl, cinnolinyl, pteridinyl, purinyl,6,7-dihydro-5H-[1]pyrindinyl, benzo[b]thiophenyl,5,6,7,8-tetrahydro-quinolin-3-yl, benzoxazolyl, benzothiazolyl,benzisothiazolyl, benzisoxazolyl, benzimidazolyl, thianaphthenyl,isothianaphthenyl, benzofuranyl, isobcnzofuranyl, isoindolyl, indolyl,indolizinyl, indazolyl, isoquinolyl, quinolyl, phthalazinyl,quinoxalinyl, quinazolinyl, benzoxazinyl; etc. One of ordinary skill inthe art will understand that the connection of said(C₂-C₉)heterocycloalkyl rings is through a carbon atom or a sp³hybridized nitrogen heteroatom.

(C₆-C₁₀)aryl when used herein refers to phenyl or naphthyl.

As used herein, the term antiviral nucleoside agent refers to antiviralnucleosides that have anti-HIV activity. The agents can be activeagainst other viral infections as well, so long as they are activeagainst HIV.

The term “antiviral thymidine nucleosides” refers to thymidine analogueswith anti-HIV activity, including but not limited to, AZT (zidovudine)and D4T (2′,3′-didehydro-3′deoxythymidine (stravudine), and 1-

-D-Dioxolane)thymine (DOT) or their prodrugs.

The term “antiviral guanine nucleosides” refers to guanine analogueswith anti-HIV activity, including but not limited to, HBG[9-(4-hydroxybutyl)guanine], lobucavir([1R(1alpha,2beta,3alpha)]-9-[2,3-bis(hydroxymethyl)cyclobutyl]guanine),abacavir((1S,4R)-4-[2-amino-6-(cyclopropylamino)-9H-purin-9-yl]-2-cyclopentene-1-methanolsulfate (salt), a prodrug of a G-carbocyclic nucleoside) and additionalantiviral guanine nucleosides disclosed in U.S. Pat. No. 5,994,321

The term “antiviral cytosine nucleosides” refers to cytosine analogueswith anti-HIV activity, including but not limited to,(−)-2′,3′-dideoxy-3′-thiacytidine (3TC) and its 5-fluoro analog[(−)-FTC, Emtricitabine], 2′,3′-dideoxycytidine (DDC), Racivir,beta-D-2′,3′-didehydro-2′,3′-dideoxy-5-fluorocytidine (DFC, D-d4FC, RVT,Dexelvucitabine) and its enantiomer L-D4FC, and apricitabine (APC,AVX754, BCH-10618).

The term “antiviral adenine nucleosides” reefers to adenine analogueswith anti-HIV activity, including, but not limited to2′,3′-dideoxy-adenosine (ddAdo), 2′,3′-dideoxyinosine (DDI),9-(2-phosphonylmethoxyethyl)adenine (PMEA), 9-R-2-phosphonomethoxypropyladenine (PMPA, Tenofovir) (K65R is resistant to PMPA), Tenofovirdisoproxil fumarate(9-[(R)-2[[bis[[isopropoxycarbonyl)oxy]-methoxy]-phosphinyl]methoxy]propyl]adeninefumarate, TDF), bis(isopropyloxymethylcarbonyl)PMPA [bis(poc)PMPA],GS-9148 (Gilead Sciences) as well as those disclosed in Balzarini, J.;De Clereq, E. Acyclic purine nucleoside phosphonates as retrovirusinhibitors. In: Jeffries D J, De Clereq E., editors. Antiviralchemotherapy. New York, N.Y: John Wiley & Sons, Inc.; 1995. pp. 41-45,the contents of which are hereby incorporated by reference.

The term AZT is used interchangeably with the term zidovudinethroughout. Similarly, abbreviated and common names for other antiviralagents are used interchangeably throughout.

As used herein, the term DAPD ((2R,4R)-2-amino-9-[(2-hydroxymethyl)-I,3-dioxolan-4-yl]adenine) is also intended to include a related form ofDAPD known as APD [(−)-β-D-2-aminopurine dioxolane], as well as alloptically active forms of DAPD, including optically active forms andracemic forms and its phosphate prodrugs as well as dioxolane-G and the6-methoxy or 6-chloro derivatives.

As used herein, the term “pharmaceutically acceptable salts” refers topharmaceutically acceptable salts which, upon administration to therecipient, are capable of providing directly or indirectly, a nucleosideantiviral agent, or that exhibit activity themselves.

As used herein, the term “prodrug,” in connection with nucleosideantiviral agents, refers to the 5′ and N-acylated, alkylated, orphosphorylated (including mono, di, and triphosphate esters as well asstabilized phosphates and phospholipid) derivatives of nucleosideantiviral agents. In one embodiment, the acyl group is a carboxylic acidester in which the non-carbonyl moiety of the ester group is selectedfrom straight, branched, or cyclic alkyl, alkoxyalkyl includingmethoxymethyl, aralkyl including benzyl, aryloxyalkyl includingphenoxymethyl, aryl including phenyl optionally substituted by halogen,alkyl, alkyl or alkoxy, sulfonate esters such as alkyl or aralkylsulphonyl including methanesulfonyl, trityl or monomethoxytrityl,substituted benzyl, trialkylsilyl, or diphenylmethylsilyl. Aryl groupsin the esters optimally comprise a phenyl group. The alkyl group can bestraight, branched or cyclic and is preferably C₁₋₁₈.

As used herein, the term “resistant virus” refers to a virus thatexhibits a three, and more typically, five or greater fold increase inEC₅₀ compared to naive virus in a constant cell line, including, but notlimited to peripheral blood mononuclear (PBM) cells, or MT2 or MT4cells.

As used herein, the term “substantially pure” or “substantially in theform of one optical isomer” refers to a composition that includes atleast 95% to 98%, or more, preferably 99% to 100%, of a singleenantiomer of the JAK inhibitors described herein, and, optionally, tosimilar concentrations of a single enantiomer of a nucleoside. In apreferred embodiment, the JAK inhibitors are administered insubstantially pure form.

I. JAK Inhibitors

Representative JAK inhibitors include those disclosed in U.S. Pat. No.7,598,257, an example of which is Ruxolitinib (Jakafi, Incyte), whichhas the structure shown below:

Representative JAK inhibitors also include those disclosed in U.S. Pat.Nos. Re 41,783; 7,842,699; 7,803,805; 7,687,507; 7,601,727; 7,569,569;7,192,963; 7,091,208; 6,890,929, 6,696,567; 6,962,993; 6,635,762;6,627,754; and 6,610,847, an example of which is Tofacitinib, which hasthe structure shown below:

Tofacitinib (Pfizer), and which has the chemical name 3-{(3R,4R)-4methyl-3-[methyl-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-amino]-piperidin-1-yl}-3-oxo-propionitrile.

In one embodiment, the compounds have the formula:

wherein:

or the pharmaceutically acceptable salt or prodrug thereof; wherein

R¹ is a group of the formula

wherein y is 0, 1 or 2;

R is selected from the group consisting of hydrogen, (C₁-C₆)alkyl,(C₁-C₆)alkylsulfonyl, (C₂-C₆)alkenyl, (C₂-C₆)alkynyl wherein the alkyl,alkenyl and alkynyl groups are optionally substituted by deuterium,hydroxy, amino, trifluoromethyl, (C₁-C₄)alkoxy, (C₁-C₆)acyloxy,(C₁-C₆)alkylamino, ((C₁-C₆)alkyl)₂amino, cyano, nitro, (C₂-C₆)alkenyl,(C₂-C₆)alkynyl or (C₁-C₆)acylamino; or

R⁴ is (C₃-C₁₀)cycloalkyl wherein the cycloalkyl group is optionallysubstituted by deuterium, hydroxy, amino, trifluoromethyl,(C₁-C₆)acyloxy, (C₁-C₆)acylamino, (C₁-C₆)alkylamino,((C₁-C₆)alkyl)₂amino, cyano, cyano(C₁-C₆)alkyl,trifluoromethyl(C₁-C₆)alkyl, nitro, nitro(C₁-C₆)alkyl or(C₁-C₆)acylamino;

R⁵ is (C₂-C₉)heterocycloalkyl wherein the heterocycloalkyl groups mustbe substituted by one to five carboxy, cyano, amino, deuterium, hydroxy,(C₁-C₆)alkyl, (C₁-C₆)alkoxy, halo, (C₁-C₆)acyl, (C₁-C₆)alkylamino,amino(C₁-C₆)alkyl, (C₁-C₆)alkoxy-CO—NH, (C₁-C₆)alkylamino-CO—,(C₂-C₆)alkenyl, (C₂-C₆) alkynyl, (C₁-C₆)alkylamino, amino(C₁-C₆)alkyl,hydroxy(C₁-C₆)alkyl, (C₁-C₆)alkoxy(C₁-C₆)alkyl,(C₁-C₆)acyloxy(C₁-C₆)alkyl, nitro, cyano(C₁-C₆)alkyl, halo(C₁-C₆)alkyl,nitro(C₁-C₆)alkyl, trifluoromethyl, trifluoromethyl(C₁-C₆)alkyl,(C₁-C₆)acylamino, (C₁-C₆)acylamino(C₁-C₆)alkyl,(C₁-C₆)alkoxy(C₁-C₆)acylamino, amino(C₁-C₆)acyl,amino(C₁-C₆)acyl(C₁-C₆)alkyl, (C₁-C₆)alkylamino(C₁-C₆)acyl,((C₁-C₆)alkyl)₂amino(C₁-C₆)acyl, R¹⁵R¹⁶N—CO—O—, R¹⁵R¹⁶N—CO—(C₁-C₆)alkyl,(C₁-C₆)alkyl-S(O)_(m), R¹⁵R¹⁶NS(O)_(m), R¹⁵R¹⁶NS(O)_(m)(C₁-C₆)alkyl,R¹⁵S(O)_(m)R¹⁶N, R¹⁵S(O)_(m)R¹⁶(C₁-C₆)alkyl wherein m is 0, 1 or 2 andR⁵ and R¹⁶ are each independently selected from hydrogen or(C₁-C₆)alkyl; or a group of the formula

wherein a is 0, 1, 2, 3 or 4;

b, c, e, f and g are each independently 0 or 1;

d is 0, 1, 2, or 3;

X is S(O)_(n) wherein n is 0, 1 or 2; oxygen, carbonyl or —C(═N-cyano)-;

Y is S(O)_(n) wherein n is 0, 1 or 2; or carbonyl; and

Z is carbonyl, C(O)O—, C(O)NR— or S(O)_(n) wherein n is 0, 1 or 2;

R⁶, R⁷, R⁸, R⁹, R¹⁰ and R¹¹ are each independently selected from thegroup consisting of hydrogen or (C₁-C₆)alkyl optionally substituted bydeuterium hydroxy, amino, trifluoromethyl, (C₁-C₆)acyloxy,(C₁-C₆)acylamino, (C₁-C₆)alkylamino, ((C₁-C₆)alkyl)₂amino, cyano,cyano(C₁-C₆)alkyl, trifluoromethyl(C₁-C₆)alkyl, nitro, nitro(C₁-C₆)alkylor (C₁-C₆)acylamino;

R¹² is carboxy, cyano, amino, oxo, deuterium, hydroxy, trifluoromethyl,(C₁-C₆)alkyl, trifluoromethyl(C₁-C₆)alkyl, (C₁-C₆)alkoxy, halo,(C₁-C₆)acyl, (C₁-C₆)alkylamino, ((C₁-C₆)alkyl)₂amino, amino(C₁-C₆)alkyl,(C₁-C₆)alkoxy-CO—NH, (C₁-C₆)alkylamino-CO—, (C₂-C₆)alkenyl, (C₂-C₆)alkynyl, (C₁-C₆)alkylamino, hydroxy(C₁-C₆)alkyl,(C₁-C₆)alkoxy(C₁-C₆)alkyl, (C₁-C₆)acyloxy(C₁-C₆)alkyl, nitro,cyano(C₁-C₆)alkyl, halo(C₁-C₆)alkyl, nitro(C₁-C₆)alkyl, trifluoromethyl,trifluoromethyl(C₁-C₆)alkyl, (C₁-C₆)acylamino,(C₁-C₆)acylamino(C₁-C₆)alkyl, (C₁-C₆)alkoxy(C₁-C₆)acylamino,amino(C₁-C₆)acyl, amino(C₁-C₆)acyl(C₁-C₆)alkyl,(C₁-C₆)alkylamino(C₁-C₆)acyl, ((C₁-C₆)alkyl)₂amino(C₁-C₆)acyl,R¹⁵R¹⁶N—CO—O—, R¹⁵R¹⁶N—CO—(C₁-C₆)alkyl, R¹⁵C(O)NH, R¹⁵OC(O)NH,R¹⁵NHC(O)NH, (C₁-C₆)alkyl-S(O)_(m), (C₁-C₆)alkyl-S(O)_(m)—(C₁-C₆)alkyl,R¹⁵R¹⁶NS(O)_(m), R¹⁵R¹⁶NS(O)(C₁-C₆)alkyl, R¹⁵S(O)_(m)R¹⁶N,R¹⁵S(O)_(m)R¹⁶N(C₁-C₆)alkyl wherein m is 0, 1 or 2 and R¹⁵ and R¹⁶ areeach independently selected from hydrogen or (C₁-C₆)alkyl;

R² and R³ are each independently selected from the group consisting ofhydrogen, deuterium, amino, halo, hydroxy, nitro, carboxy,(C₂-C₆)alkenyl, (C₂-C₆)alkynyl, trifluoromethyl, trifluoromethoxy,(C₁-C₆)alkyl, (C₁-C₆)alkoxy, (C₃-C₁₀)cycloalkyl wherein the alkyl,alkoxy or cycloalkyl groups are optionally substituted by one to threegroups selected from halo, hydroxy, carboxy, amino (C₁-C₆)alkylthio,(C₁-C₆)alkylamino, ((C₁-C₆)alkyl)₂amino, (C₅-C₉)heteroaryl,(C₂-C₉)heterocycloalkyl, (C₃-C₉)cycloalkyl or (C₆-C₁₀)aryl; or R² and R³are each independently (C₃-C₁₀)cycloalkyl, (C₃-C₁₀)cycloalkoxy,(C₁-C₆)alkylamino, ((C₁-C₆)alkyl)₂amino, (C₆-C₁₀)arylamino,(C₁-C₆)alkylthio, (C₆-C₁₀)arylthio, (C₁-C₆)alkylsulfinyl,(C₆-C₁₀)arylsulfinyl, (C₁-C₆)alkylsulfonyl, (C₆-C₁₀)arylsulfonyl,(C₁-C₆)acyl, (C₁-C₆)alkoxy-CO—NH—, (C₁-C₆)alkylamino-CO—,(C₅-C₉)heteroaryl, (C₂-C₉)heterocycloalkyl or (C₆-C₁₀)aryl wherein theheteroaryl, heterocycloalkyl and aryl groups are optionally substitutedby one to three halo, (C₁-C₆)alkyl, (C₁-C₆)alkyl-CO—NH—,(C₁-C₆)alkoxy-CO—NH—, (C₁-C₆)alkyl-CO—NH—(C₁-C₆)alkyl,(C₁-C₆)alkoxy-CO—NH—(C₁-C₆)alkyl, (C₁-C₆)alkoxy-CO—NH—(C₁-C₆)alkoxy,carboxy, carboxy(C₁-C₆)alkyl, carboxy(C₁-C₆)alkoxy,benzyloxycarbonyl(C₁-C₆)alkoxy, (C₁-C₆)alkoxycarbonyl(C₁-C₆)alkoxy,(C₆-C₁₀)aryl, amino, amino(C₁-C₆)alkyl, (C₁-C₆)alkoxycarbonylamino,(C₆-C₁₀)aryl(C₁-C₆)alkoxycarbonylamino, (C₁-C₆)alkylamino,((C₁-C₆)alkyl)₂amino, (C₁-C₆)alkylamino(C₁-C₆)alkyl,((C₁-C₆)alkyl)₂amino(C₁-C₆)alkyl, hydroxy, (C₁-C₆)alkoxy, carboxy,carboxy(C₁-C₆)alkyl, (C₁-C₆)alkoxycarbonyl,(C₁-C₆)alkoxycarbonyl(C₁-C₆)alkyl, (C₁-C₆)alkoxy-CO—NH—,(C₁-C₆)alkyl-CO—NH—, cyano, (C₅-C₉)heterocycloalkyl, amino-CO—NH—,(C₁-C₆)alkylamino-CO—NH—, ((C₁-C₆)alkyl)₂amino-CO—NH—,(C₆-C₁₀)arylamino-CO—NH—, (C₅-C₉)heteroarylamino-CO—NH—,(C₁-C₆)alkylamino-CO—NH—(C₁-C₆)alkyl,((C₁-C₆)alkyl)₂amino-CO—NH—(C₁-C₆)alkyl,(C₆-C₁₀)arylamino-CO—NH—(C₁-C₆)alkyl,(C₅-C₉)heteroarylamino-CO—NH—(C₁-C₆)alkyl, (C₁-C₆)alkylsulfonyl,(C₁-C₆)alkylsulfonylamino, (C₁-C₆)alkylsulfonylamino(C₁-C₆)alkyl,(C₆-C₁₀)arylsulfonyl, (C₆-C₁₀)arylsulfonylamino,(C₆-C₁₀)arylsulfonylamino(C₁-C₆)alkyl, (C₁-C₆)alkylsulfonylamino,(C₁-C₆)alkylsulfonylamino(C₁-C₆)alkyl, (C₅-C₉)heteroaryl or(C₂-C₉)heterocycloalkyl.

The JAK inhibitors also include compounds of Formula B:

including pharmaceutically acceptable salt forms or prodrugs thereof,wherein:

A¹ and A² are independently selected from C and N;

T, U, and V are independently selected from O, S, N, CR⁵, and NR⁶;

wherein the 5-membered ring formed by A¹, A², U, T, and V is aromatic;

X is N or CR⁴;

Y is C₁₋₈ alkylene, C₂₋₈ alkenylene, C₂₋₈ alkynylene,(CR¹¹R¹²)_(p)-(C₃₋₁₀ cycloalkylene)-(CR¹¹R¹²)_(q),(CR¹¹R¹²)_(p)-(arylene)-(CR¹¹R¹²)_(q), (CR¹¹R¹²)_(p)-(C₁₋₁₀heterocycloalkylene)-(CR¹¹R¹²)_(q),(CR¹¹R¹²)_(p)-(heteroarylene)-(CR¹¹R¹²)_(q),(CR¹¹R¹²)_(p)O(CR¹¹R¹²)_(q), (CR¹¹R¹²)_(p)S(CR¹¹R¹²)_(q),(CR¹¹R¹²)_(p)C(O)(CR¹¹R¹²)_(q), (CR¹¹R¹²)_(p)C(O)NR_(c)(CR¹¹R¹²)_(q),(CR¹¹R¹²)_(p)C(O)O(CR¹¹R¹²)_(q), (CR¹¹R¹²)_(p)OC(O)(CR¹¹R¹²)_(q),(CR¹¹R¹²)_(p)OC(O)NR^(c)(CR¹¹R¹²)_(q), (CR¹¹R¹²)_(p)NR^(c)(CR¹¹R¹²)_(q),(CR¹¹R¹²)_(p)NR^(c)C(O)NR^(d)(CR¹¹R¹²)_(q),(CR¹¹R¹²)_(p)S(O)(CR¹¹R¹²)_(q), (CR¹¹R¹²)_(p)S(O)NR^(c)(CR¹¹R¹²)_(q).(CR¹¹R¹²)_(p)S(O)₂(CR¹¹R¹²)_(q), or(CR¹¹R¹²)_(p)S(O)₂NR^(c)(CR¹¹R¹²)_(q), wherein said C₁₋₈ alkylene, C₂₋₈alkenylene, C₂₋₈ alkynylene, cycloalkylene, arylene,heterocycloalkylene, or heteroarylene, is optionally substituted with 1,2, or 3 substituents independently selected from -D¹-D²-D³-D⁴;

Z is H, halo, C₁₋₄ alkyl, C₂₋₄ alkenyl, C₂₋₄ alkynyl, C₁₋₄ haloalkyl,halosulfanyl, C₁₋₄ hydroxyalkyl, C₁₋₄ cyanoalkyl, ═C—R^(i), ═N—R^(i),Cy¹, CN, NO₂, OR^(a), SR^(a), C(O)R^(b), C(O)NR^(c)R^(d), C(O)OR^(a),OC(O)R^(b), OC(O)NR^(c)R^(d), NR^(c)R^(d), NR^(c)C(O)R^(b),NR^(c)C(O)NR^(c)R^(d), NR^(c)C(O)OR^(a), C(═NR^(i))NR^(c)R^(d),NR^(c)C(═NR^(i))NR^(c)R^(d), S(O)R^(b), S(O)NR^(c)R^(d). S(O)₂R^(b),NR^(c)S(O)₂R^(b), C(═NOH)R^(b), C(═NO(C₁₋₆alkyl)R^(b), andS(O)₂NR^(c)R^(d), wherein said C₁₋₈ alkyl, C₂₋₈ alkenyl, or C₂₋₈alkynyl, is optionally substituted with 1, 2, 3, 4, 5, or 6 substituentsindependently selected from halo, C₁₋₄ alkyl, C₂₋₄ alkenyl, C₂₋₄alkynyl, C₁₋₄ haloalkyl, halosulfanyl, C₁₋₄ hydroxyalkyl, C₁₋₄cyanoalkyl, Cy¹, CN, N₂, OR^(a), SR^(a), C(O)R^(b), C(O)NR^(c)R^(d),C(O)OR^(a), OC(O)R^(b), OC(O)NR^(c)R^(d), NR^(c)R^(d), NR^(c)C(O)R^(b),NR^(c)C(O)NR^(c)R^(d), NR^(c)C(O)OR^(a), C(═NR^(i))NR^(c)R^(d),NR^(c)C(═NR^(i))NR^(c)R^(d), S(O)R^(b), S(O)NR^(c)R^(d), S(O)₂R,NR^(c)S(O)₂R^(b), C(═NOH)R^(b), C(═NO(C₁₋₆ alkyl))R^(b), andS(O)₂NR^(c)R^(d);

wherein when Z is H, n is 1;

or the —(Y)_(n)—Z moiety is taken together with i) A² to which themoiety is attached, ii) R⁵ or R⁶ of either T or V, and iii) the C or Natom to which the R⁵ or R⁶ of either T or V is attached to form a 4- to20-membered aryl, cycloalkyl, heteroaryl, or heterocycloalkyl ring fusedto the 5-membered ring formed by A¹ A², U, T, and V, wherein said 4- to20-membered aryl, cycloalkyl, heteroaryl, or heterocycloalkyl ring isoptionally substituted by 1, 2, 3, 4, or 5 substituents independentlyselected from —(W)_(m)-Q;

W is C₁₋₈ alkylenyl, C₂₋₈ alkenylenyl, C₂₋₈ alkynylenyl, O, S, C(O),C(O)NR^(c′), C(O)O, OC(O), OC(O)NR^(c′), NR^(c′), NR^(c′)C(O)NR^(d′),S(O), S(O)NR^(c′), S(O)₂, or S(O)₂NR^(c′);

Q is H, halo, CN, NO₂, C₁₋₈ alkyl, C₂₋₈ alkenyl, C₂₋₈ alkynyl, C₁₋₈haloalkyl, halosulfanyl, aryl, cycloalkyl, heteroaryl, orheterocycloalkyl, wherein said C₁₋₈ alkyl, C₂₋₈ alkenyl, C₂₋₈ alkynyl,C₁₋₈ haloalkyl, aryl, cycloalkyl, heteroaryl, or heterocycloalkyl isoptionally substituted with 1, 2, 3 or 4 substituents independentlyselected from halo, C₁₋₄ alkyl, C₂₋₄ alkenyl, C₂₋₄ alkynyl, C₁₋₄haloalkyl, halosulfanyl, C₁₋₄ hydroxyalkyl, C₁₋₄ cyanoalkyl, Cy², CN,NO₂, OR^(a′), SR^(a′), C(O)R^(b′), C(O)NR^(c′)R^(d′), C(O)OR^(a′),OC(O)R^(b′), OC(O)NR^(c′)R^(d′), NR^(c′)R^(d′), NR^(c′)C(O)R^(b′),NR^(c′)C(O)N R^(c′)R^(d′), N R^(c′)C(O)OR^(a′), S(O)R^(b′), S(O)NR^(c′)R^(d′), S(O)₂R^(b′), NR^(c)′S(O)₂R^(b′), and S(O)₂N R^(c′)R^(d′);

Cy¹ and Cy² are independently selected from aryl, heteroaryl,cycloalkyl, and heterocycloalkyl, each optionally substituted by 1, 2,3, 4 or 5 substituents independently selected from halo, C₁₋₄ alkyl,C₂₋₄ alkenyl, C₂₋₄ alkynyl, C₁₋₄ haloalkyl, halosulfanyl, C₁₋₄hydroxyalkyl, C₁₋₄ cyanoalkyl, CN, NO₂, OR^(a″), SR^(a″), C(O)R^(b)″,C(O)NR^(c′)′R^(d″), C(O)OR^(a″), OC(O)R^(b″), OC(O)N R^(c′)′R^(d″),NR^(c′)′R^(d″), NR^(c′)′C(O)R^(b″), NR^(c″)C(O)OR^(a″),NR^(c″)S(O)R^(b″), NR^(c″)S(O)₂R^(b″), S(O)R^(b)″, S(O)NR^(c″)R^(d″),S(O)₂R^(b″), and S(O)₂NR^(c″)R^(d″);

R¹, R², R³, and R⁴ are independently selected from H, halo, C₁₋₄ alkyl,C₂₋₄ alkenyl, C₂₋₄ alkynyl, C₁₋₄ haloalkyl, halosulfanyl, aryl,cycloalkyl, heteroaryl, heterocycloalkyl, CN, NO₂, OR⁷, SR⁷, C(O)R⁸,C(O)NR⁹R¹⁰, C(O)OR⁷OC(O)R⁸, OC(O)NR⁹R¹⁰, NR⁹R¹⁰, NR⁹C(O)R⁸,NR^(c)C(O)OR⁷, S(O)R⁸, S(O)NR⁹R¹⁰, S(O)₂R⁸, NR⁹S(O)₂R⁸, and S(O)₂NR⁹R¹⁰.

R⁵ is H, halo, C₁₋₄ alkyl, C₂₋₄ alkenyl, C₂₋₄ alkynyl, C₁₋₄ haloalkyl,halosulfanyl, CN, NO₂, OR⁷, SR⁷, C(O)R⁸, C(O)NR⁹R¹⁰, C(O)OR⁷, OC(O)R⁸,OC(O)NR⁹R¹⁰, NR⁹R¹⁰, NR⁹C(O)R⁸, NR⁹C(O)OR⁷, S(O)R⁸, S(O)NR⁹R¹⁰, S(O)₂R⁸,NR⁹S(O)₂R⁸, or S(O)₂NR⁹R¹⁰;

R⁶ is H, C₁₋₄ alkyl, C₂₋₄ alkenyl, C₂₋₄ alkynyl, C₁₋₄ haloalkyl, OR⁷,C(O)R⁸, C(O)NR⁹R¹⁰, C(O)OR⁷, S(O)R⁸, S(O)NR⁹R¹⁰, S(O)₂R⁸, orS(O)₂NR⁹R¹⁰;

R⁷ is H, C₁₋₆ alkyl, C₁₋₆ haloalkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, aryl,cycloalkyl, heteroaryl, heterocycloalkyl, arylalkyl, heteroarylalkyl,cycloalkylalkyl or heterocycloalkylalkyl;

R⁸ is H, C₁₋₄ alkyl, C₁₋₄ haloalkyl, C₂₋₄ alkenyl, C₂₋₆ alkynyl, aryl,cycloalkyl, heteroaryl, heterocycloalkyl, arylalkyl, heteroarylalkyl,cycloalkylalkyl or heterocycloalkylalkyl;

R⁹ and R¹⁰ are independently selected from H, C₁₋₁₀ alkyl, C₁₋₆haloalkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₁₋₆ alkylcarbonyl, arylcarbonyl,C₁₋₆ alkylsulfonyl, arylsulfonyl, aryl, heteroaryl, cycloalkyl,heterocycloalkyl, arylalkyl, heteroarylalkyl, cycloalkylalkyl andheterocycloalkylalkyl;

or R⁹ and R¹⁰ together with the N atom to which they are attached form a4-, 5-, 6- or 7-membered heterocycloalkyl group;

R¹¹ and R¹² are independently selected from H and -E¹-E²-E³-E⁴;

D¹ and E¹ are independently absent or independently selected from C₁₋₄alkylene, C₂₋₆ alkenylene, C₂₋₆ alkynylene, arylene, cycloalkylene,heteroarylene, and heterocycloalkylene, wherein each of the C₁₋₆alkylene, C₂₋₆ alkenylene, C₂₋₆ alkynylene, arylene, cycloalkylene,heteroarylene, and heterocycloalkylene is optionally substituted by 1, 2or 3 substituents independently selected from halo, CN, NO₂, N₃, SCN,OH, C₁₋₄ alkyl, C₁₋₄ haloalkyl, C₂₋₈ alkoxyalkyl, C₁₋₄ alkoxy, C₁₋₄haloalkoxy, amino, C₁₋₄ alkylamino, and C₂₋₈ dialkylamino;

D² and E² are independently absent or independently selected from C₁₋₆alkylene, C₂₋₄ alkenylene, C₂₋₄ alkynylene, (C₁₋₄ alkylene)_(r)-O—(C₁₋₆alkylene)_(s), (C₁₋₆ alkylene)_(r)-S—(C₁₋₆ alkylene)_(s), (C₁₋₆alkylene)_(r)-NR^(c)—(C₁₋₆ alkylene)_(s), (C₁₋₆ alkylene)_(r)-CO—(C₁₋₆alkylene)_(s), (C₁₋₆ alkylene)_(r)-COO—(C₁₋₆ alkylene)_(s), (C₁₋₆alkylene)_(r)-CONR^(c)—(C₁₋₆ alkylene)_(s), (C₁₋₆ alkylene)_(r)-SO—(C₁₋₆alkylene)_(s), (C₁₋₆ alkylene)_(r)-SO₂—(C₁₋₄ alkylene)_(s), (C₁₋₆alkylene)_(r)-SONR^(c)C—(C₁₋₆ alkylene)_(s), and (C₁₋₆alkylene)_(r)-NR^(e)CONR^(f)—(C₁₋₆ alkylene)_(s), wherein each of theC₁₋₆ alkylene, C₂₋₆ alkenylene, and C₂₋₆ alkynylene is optionallysubstituted by 1, 2 or 3 substituents independently selected from halo,CN, NO₂, N₃, SCN, OH, C₁₋₆ alkyl, C₁₋₆ haloalkyl, C₂₋₈ alkoxyalkyl, C₁₋₆alkoxy, C₁₋₆ haloalkoxy, amino, C₁₋₆ alkylamino, and C₂₋₈ dialkylamino;

D³ and E³ are independently absent or independently selected from C₁₋₆alkylene, C₂₋₆ alkenylene, C₂₋₆ alkynylene, arylene, cycloalkylene,heteroarylene, and heterocycloalkylene, wherein each of the C₁₋₆alkylene, C₂₋₆ alkenylene, C₂₋₆ alkynylene, arylene, cycloalkylene,heteroarylene, and heterocycloalkylene is optionally substituted by 1, 2or 3 substituents independently selected from halo, CN, NO₂, N₃, SCN,OH, C₁₋₆ alkyl, C₁₋₆ haloalkyl, C₂₋₈ alkoxyalkyl, C₁₋₆ alkoxy, C₁₋₆haloalkoxy, amino, C₁₋₆ alkylamino, and C₂₋₈ dialkylamino;

E⁴ and E⁴ are independently selected from H, halo, C₁₋₄ alkyl, C₂₋₄alkenyl, C₂₋₄ alkynyl, C₁₋₄ haloalkyl, halosulfanyl, C₁₋₄ hydroxyalkyl,C₁₋₄ cyanoalkyl, Cy¹, CN, NO₂, OR^(a), SR^(a), C(O)R^(b),C(O)NR^(c)R^(d), C(O)OR^(a), OC(O)R^(b)OC(O)NR^(c)R^(d) NR^(c)R^(d),NR^(c)C(O)R^(b), NR^(c)C(O)NR^(c)R^(d), NR^(c)C(O)OR^(a),C(═NR^(i))NR^(c)R^(d), NR^(c)C(═NR^(i))NR^(c)R^(d), S(O)R^(b),S(O)NR^(c)R^(d), S(O)₂R^(b), NR^(c)S(O)₂R^(b), C(═NOH)R^(b), C(═NO(C₁₋₆alkyl)R^(b), and S(O)₂NR^(c)R^(d), wherein said C₁₋₈ alkyl, C₂₋₈alkenyl, or C₂₋₈ alkynyl, is optionally substituted with 1, 2, 3, 4, 5,or 6 substituents independently selected from halo, C₁₋₄ alkyl, C₂₋₄alkenyl, C₂₋₄ alkynyl, C₁₋₄ haloalkyl, halosulfanyl, C₁₋₄ hydroxyalkyl,C₁₋₄ cyanoalkyl, Cy¹, CN, NO₂, OR^(a), SR^(a), C(O)R^(b),C(O)NR^(c)R^(d), C(O)OR^(a), OC(O)R^(b), OC(O)NR^(c)R^(d), NR^(c)R^(d),NR^(c)C(O)R^(b), NR^(c)C(O)NR^(c)R^(d), NR^(c)C(O)OR^(a),C(═NR^(i))NR^(c)R^(d), NR^(c)C(═NR^(i))NR^(c)R^(d), S(O)R^(b),S(O)NR^(c)R^(d), S(O)₂R^(b), NR^(c)S(O)₂R^(b), C(═NOH)R^(b), C(═NO(C₁₋₆alkyl))R^(b), and S(O)₂NR^(c)R^(d);

R^(a) is H, Cy¹, —(C₁₋₆ alkyl)-Cy¹, C₁₋₆ alkyl, C₁₋₆ haloalkyl, C₂₋₆alkenyl, C₂₋₆ alkynyl, wherein said C₁₋₆ alkyl, C₁₋₆ haloalkyl, C₂₋₆alkenyl, or C₂₋₆ alkynyl is optionally substituted with 1, 2, or 3substituents independently selected from OH, CN, amino, halo, C₁₋₄alkyl, C₁₋₄ haloalkyl, halosulfanyl, aryl, arylalkyl, heteroaryl,heteroarylalkyl, cycloalkyl and heterocycloalkyl;

R^(b) is H, Cy¹, —(C₁₋₄ alkyl)-Cy¹, C₁₋₆ alkyl, C₁₋₆ haloalkyl, C₂₋₆alkenyl, C₂₋₆ alkynyl, wherein said C₁₋₆ alkyl, C₁₋₆₁₋₆ haloalkyl, C₂₋₆alkenyl, or C₂₋₆ alkynyl is optionally substituted with 1, 2, or 3substituents independently selected from OH, CN, amino, halo, C₁₋₆alkyl, C₁₋₆ haloalkyl, C₁₋₆ haloalkyl, halosulfanyl, aryl, arylalkyl,heteroaryl, heteroarylalkyl, cycloalkyl and heterocycloalkyl;

R^(a′) and R^(a″) are independently selected from H, C₁₋₆ alkyl, C₁₋₆haloalkyl, C₂₋₆ alkenyl, C₂₋₄ alkynyl, aryl, cycloalkyl, heteroaryl,heterocycloalkyl, arylalkyl, heteroarylalkyl, cycloalkylalkyl andheterocycloalkylalkyl, wherein said C₁₋₈ alkyl, C₁₋₆ haloalkyl, C₂₋₆alkenyl, C₂₋₆ alkynyl, aryl, cycloalkyl, heteroaryl, heterocycloalkyl,arylalkyl, heteroarylalkyl, cycloalkylalkyl or heterocycloalkylalkyl isoptionally substituted with 1, 2, or 3 substituents independentlyselected from OH, CN, amino, halo, C₁₋₆ alkyl, C₁₋₆ haloalkyl,halosulfanyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, cycloalkyland heterocycloalkyl;

R^(b′) and R^(b″) are independently selected from H, C₁₋₆ alkyl, C₁₋₆haloalkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, aryl, cycloalkyl, heteroaryl,heterocycloalkyl, arylalkyl, heteroarylalkyl, cycloalkylalkyl andheterocycloalkylalkyl, wherein said C₁₋₆ alkyl, C₁₋₆ haloalkyl, C₂₋₆alkenyl, C₂₋₆ alkynyl, aryl, cycloalkyl, heteroaryl, heterocycloalkyl,arylalkyl, heteroarylalkyl, cycloalkylalkyl or heterocycloalkylalkyl isoptionally substituted with 1, 2, or 3 substituents independentlyselected from OH, CN, amino, halo, C₁₋₆ alkyl, C₁₋₆ haloalkyl, C₁₋₆haloalkyl, halosulfanyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl,cycloalkyl and heterocycloalkyl;

R^(c) and R^(d) are independently selected from H, Cy¹, —(C₁₋₆alkyl)-Cy¹, C₁₋₁₀ alkyl, C₁₋₆ haloalkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl,wherein said C₁₋₁₀ alkyl, C₁₋₆ haloalkyl, C₂₋₆ alkenyl, or C₂₋₆ alkynyl,is optionally substituted with 1, 2, or 3 substituents independentlyselected from Cy¹, —(C₁₋₄ alkyl)-Cy¹, OH, CN, amino, halo, C₁₋₆ alkyl,C₁₋₆ haloalkyl, C₁₋₆ haloalkyl, and halosulfanyl;

or R^(c) and R^(d) together with the N atom to which they are attachedform a 4-, 5-, 6- or 7-membered heterocycloalkyl group optionallysubstituted with 1, 2, or 3 substituents independently selected fromCy¹, —(C₁₋₄ alkyl)-Cy¹, OH, CN, amino, halo, C₁₋₆ alkyl, C₁₋₆ haloalkyl,C₁₋₄ haloalkyl, and halosulfanyl;

R^(c)′ and R^(d)′ are independently selected from H, C₁₋₁₀ alkyl, C₁₋₆haloalkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, aryl, heteroaryl, cycloalkyl,heterocycloalkyl, arylalkyl, heteroarylalkyl, cycloalkylalkyl andheterocycloalkylalkyl, wherein said C₁₋₁₀ alkyl, C₁₋₆ haloalkyl, C₂₋₆alkenyl, C₂₋₆ alkynyl, aryl, heteroaryl, cycloalkyl, heterocycloalkyl,arylalkyl, heteroarylalkyl, cycloalkylalkyl or heterocycloalkylalkyl isoptionally substituted with 1, 2, or 3 substituents independentlyselected from OH, CN, amino, halo, C₁₋₆ alkyl, C₁₋₆ haloalkyl, C₁₋₆haloalkyl, halosulfanyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl,cycloalkyl and heterocycloalkyl;

or R^(c)′ and R^(d)′ together with the N atom to which they are attachedform a 4-, 5-, 6- or 7-membered heterocycloalkyl group optionallysubstituted with 1, 2, or 3 substituents independently selected from OH,CN, amino, halo, C₁₋₆ alkyl, C₁₋₆ haloalkyl, C₁₋₆ haloalkyl,halosulfanyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, cycloalkyland heterocycloalkyl;

R^(c)″ and R^(d)″ are independently selected from H, C₁₋₁₀ alkyl, C₁₋₆haloalkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, aryl, heteroaryl, cycloalkyl,heterocycloalkyl, arylalkyl, heteroarylalkyl, cycloalkylalkyl andheterocycloalkylalkyl, wherein said C₁₋₁₀ alkyl, C₁₋₆ haloalkyl, C₂₋₆alkenyl, C₂₋₆ alkynyl, aryl, heteroaryl, cycloalkyl, heterocycloalkyl,arylalkyl, heteroarylalkyl, cycloalkylalkyl or heterocycloalkylalkyl isoptionally substituted with 1, 2, or 3 substituents independentlyselected from OH, CN, amino, halo, C₁₋₆ alkyl, C₁₋₆ haloalkyl,halosulfanyl, C₁₋₆ haloalkyl, aryl, arylalkyl, heteroaryl,heteroarylalkyl, cycloalkyl and heterocycloalkyl;

or R^(c)″ and R^(d)″ together with the N atom to which they are attachedform a 4-, 5-, 6- or 7-membered heterocycloalkyl group optionallysubstituted with 1, 2, or 3 substituents independently selected from OH,CN, amino, halo, C₁₋₆ alkyl, C₁₋₆ haloalkyl, C₁₋₆ haloalkyl,halosulfanyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, cycloalkyland heterocycloalkyl;

R^(i) is H, CN, NO₂, or C₁₋₄ alkyl;

R^(e) and R^(f) are independently selected from H and C₁₋₆ alkyl;

R^(i) is H, CN, or NO₂;

m is 0 or 1;

n is 0 or 1;

p is 0, 1, 2, 3, 4, 5, or 6;

q is 0, 1, 2, 3, 4, 5 or 6;

r is 0 or 1; and

s is 0 or 1.

Additional JAK inhibitors include CEP-701 (Lestaurtinib, CephalonTechnology), a JAK 2 FL3 kinase, AZD1480 (Astra Zeneca), a JAK 2inhibitor, LY3009104/INCB28050 (Eli Lilly, Incyte), a JAK 1/2 inhibitor,Pacritinib/SB1518 (S*BIO), a JAK 2 inhibitor, VX-509 (Vertex), a JAK 3inhibitor, GLPG0634 (Galapagos), a JAK 1 inhibitor, INC424 (Novartis), aJAK inhibitor, R-348 (Rigel), a JAK 3 inhibitor, CYT387 (YM Bioscience),a JAK1/2 inhibitor, TG 10138, a JAK 2 inhibitor, AEG 3482 (Axon), a JAKinhibitor, and pharmaceutically-acceptable salts and prodrugs thereof.

Lestaurtinib has the following formula:

AEG 3482 has the following formula:

TG 10138 has the following formula:

CYT387 has the following formula:

AZD1480 has the following formula:

LY3009104 is believed to be(R)-3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl-3-cyclopentyl-propanenitrile

Pacritinib has the following formula:

The compounds include those described in U.S. Publication Nos.20110020469; 20110118255; 20100311743; 20100310675; 20100280026;20100160287; 20100081657; 20100081645; 20090181938; 20080032963;20070259869; and 20070249031.

The compounds also include those described in U.S. Publication Nos.20110251215; 20110224157; 20110223210; 20110207754; 20110136781;20110086835; 20110086810; 20110082159; 20100190804; 20100022522;20090318405; 20090286778; 20090233903; 20090215766; 20090197869;20090181959; 20080312259; 20080312258; 20080188500; and 20080167287;20080039457.

The compounds also include those described in U.S. Publication Nos.20100311693; 20080021013; 20060128780; 20040186157; and 20030162775.

The compounds also include those described in U.S. Publication Nos.20110245256; 20100009978; 20090098137; and 20080261973.

The compounds also include those described in U.S. Publication No.20110092499. Representative compounds include:

1. 7-iodo-N-(4-morpholinophenyl)thieno[3,2-d]pyrimidin-2-amine 2.7-(4-aminophenyl)-N-(4-morpholinophenyl)thieno[3,2-d]pyrimidin-2-amine3.N-(4-(2-(4-morpholinophenylamino)thieno[3,2-d]pyrimidin-7-yl)phenyl)acryl-amide4.7-(3-aminophenyl)-N-(4-morpholinophenyl)thieno[3,2-d]pyrimidin-2-amine5.N-(3-(2-(4-morpholinophenylamino)thieno[3,2-d]pyrimidin-7-yl)phen-yl)acrylamide7. N-(4-morpholinophenyl)thieno[3,2-d]pyrimidin-2-amine 8. methyl2-(4-morpholinophenylamino)thieno[3,2-d]pyrimidine-7-carboxylate 9.N-(4-morpholinophenyl)-5H-pyrrolo[3,2-d]pyrimidin-2-amine 10.7-(4-amino-3-methoxyphenyl)-N-(4-morpholinophenyl)thieno[3,2-d]pyrimidin-2-amine11.4-(2-(4-morpholinophenylamino)thieno[3,2-d]pyrimidin-7-yl)benzenesulfonam-ide12.N,N-dimethyl-3-(2-(4-morpholinophenylamino)thieno[3,2-d]pyrimidin-7-yl)benzenesulfonamide13.1-ethyl-3-(2-methoxy-4-(2-(4-morpholinophenylamino)thieno[3,2-d]pyrimidin-7-yl)phenyl)urea14.N-(4-(2-(4-morpholinophenylamino)thieno[3,2-d]pyrimidin-7-yl)phenyl)metha-nesulfonamide15.2-methoxy-4-(2-(4-morpholinophenylamino)thieno[3,2-d]pyrimidin-7-yl)pheno-l16.2-cyano-N-(3-(2-(4-morpholinophenylamino)thieno[3,2-d]pyrimidin-7-yl-)phenyl)acetamide17.N-(cyanomethyl)-2-(4-morpholinophenylamino)thieno[3,2-d]pyrimidine-7-carb-oxamide18.N-(3-(2-(4-morpholinophenylamino)thieno[3,2-d]pyrimidin-7-yl)phenyl)metha-nesulfonamide19.1-ethyl-3-(4-(2-(4-morpholinophenylamino)thieno[3,2-d]pyrimidin-7-yl)-2-(-trifluoromethoxy)phenyl)urea20. N-(3-nitrophenyl)-7-phenylthieno[3,2-d]pyrimidin-2-amine 21.7-iodo-N-(3-nitrophenyl)thieno[3,2-d]pyrimidin-2-amine 22.N1-(7-(2-ethylphenyl)thieno[3,2-d]pyrimidin-2-yl)benzene-1,3-diamine 25.N-tert-butyl-3-(2-(4-morpholinophenylamino)thieno[3,2-d]pyrimidin-7-yl)be-nzenesulfonamide26. N1-(7-iodothieno[3,2-d]pyrimidin-2-yl)benzene-1,3-diamine 28.7-(4-amino-3-(trifluoromethoxy)phenyl)-N-(4-morpholinophenyl)thieno[3,2-d]pyrimidin-2-amin-e29.7-(2-ethylphenyl)-N-(4-morpholinophenyl)thieno[3,2-d]pyrimidin-2-ami-ne30.N-(3-(2-(4-morpholinophenylamino)thieno[3,2-d]pyrimidin-7-yl)phenyl-)acetamide31.N-(cyanomethyl)-N-(3-(2-(4-morpholinophenylamino)thieno[3,2-d]pyrimidin-7-yl)phenyl)methanesulfonamide32.N-(cyanomethyl)-N-(4-(2-(4-morpholinophenylamino)thieno[3,2-d]pyrimidin-7-yl)phenyl)methanesulfonamide33.N-(3-(5-methyl-2-(4-morpholinophenylamino)-5H-pyrrolo[3,2-d]pyrimidin-7-y-l)phenyl)methanesulfonamide34.4-(5-methyl-2-(4-morpholinophenylamino)-5H-pyrrolo[3,2-d]pyrimidin-7-yl)b-enzenesulfonamide36.N-(4-(5-methyl-2-(4-morpholinophenylamino)-5H-pyrrolo[3,2-d]pyrimidin-7-y-l)phenyl)methanesulfonamide37. 7-iodo-N-(4-morpholinophenyl)-5H-pyrrolo[3,2-d]pyrimidin-2-amine 38.7-(2-isopropylphenyl)-N-(4-morpholinophenyl)thieno[3,2-d]pyrimidin-2-amin-e39. 7-bromo-N-(4-morpholinophenyl)thieno[3,2-d]pyrimidin-2-amine 40.N7-(2-isopropylphenyl)-N2-(4-morpholinophenyl)thieno[3,2-d]pyrimidine-2,7-diamine41.N7-(4-isopropylphenyl)-N2-(4-morpholinophenyl)thieno[3,2-d]pyrimidine-2,7-diamine42.7-(5-amino-2-methylphenyl)-N-(4-morpholinophenyl)thieno[3,2-d]pyrimidin-2-amine43.N-(cyanomethyl)-4-(2-(4-morpholinophenylamino)thieno[3,2-d]pyri-midin-7-yl)benzamide44. 7-iodo-N-(3-morpholinophenyl)thieno[3,2-d]pyrimidin-2-amine 45.7-(4-amino-3-nitrophenyl)-N-(4-morpholinophenyl)thieno[3,2-d]pyrimidin-2-amine46.7-(2-methoxypyridin-3-yl)-N-(4-morpholinophenyl)thieno[3,2-d]pyr-imidin-2-amine47. (3-(7-iodothieno[3,2-d]pyrimidin-2-ylamino)phenyl)methanol 48.N-tert-butyl-3-(2-(3-morpholinophenylamino)thieno[3,2-d]pyrimidin-7-yl)be-nzenesulfonamide49.N-tert-butyl-3-(2-(3-(hydroxymethyl)phenylamino)thieno[3,2-d]pyrimidin-7-yl)benzenesulfonamide50.N-(4-morpholinophenyl)-7-(4-nitrophenylthio)-5H-pyrrolo[3,2-d]pyrimidin-2-amine51.N-tert-butyl-3-(2-(3,4,5-trimethoxyphenylamino)thieno[3,2-d]pyr-imidin-7-yl)benzenesulfonamide52.7-(4-amino-3-nitrophenyl)-N-(3,4-dimethoxyphenyl)thieno[3,2-d]pyrimidin-2-amine53.N-(3,4-dimethoxyphenyl)-7-(2-methoxypyridin-3-yl)thieno[3,2-d]p-yrimidin-2-amine54.N-tert-butyl-3-(2-(3,4-dimethoxyphenylamino)thieno[3,2-d]pyrimidin-7-yl)b-enzenesulfonamide55.7-(2-aminopyrimidin-5-yl)-N-(3,4-dimethoxyphenyl)thieno[3,2-d]pyrimidin-2-amine56.N-(3,4-dimethoxyphenyl)-7-(2,6-dimethoxypyridin-3-yl)thieno[3,2-d]pyrimidin-2-amine57.N-(3,4-dimethoxyphenyl)-7-(2,4-dimethoxypyrimidin-5-yl)thieno[3,2-d]pyrim-idin-2-amine10 58.7-iodo-N-(4-(morpholinomethyl)phenyl)thieno[3,2-d]pyrimidin-2-amine 59.N-tert-butyl-3-(2-(4-(morpholinomethyl)phenylamino)thieno[3,2-d]pyrimidin-7-yl)benzenesulfonamide60.2-cyano-N-(4-methyl-3-(2-(4-morpholinophenylamino)thieno[3,2-d]pyrimidin-7-yl)phenyl)acetamide61. ethyl3-(2-(4-morpholinophenylamino)thieno[3,2-d]pyrimidin-7-yl)benzoate 62.7-bromo-N-(4-(2-(pyrrolidin-1-yl)ethoxy)phenyl)thieno[3,2-d]pyrimidin-2-a-mine63.N-(3-(2-(4-(2-(pyrrolidin-1-yl)ethoxy)phenylamino)thieno[3,2-d]py-rimidin-7-yl)phenyl)acetamide64.N-(cyanomethyl)-3-(2-(4-morpholinophenylamino)thieno[3,2-d]pyrimidin-7-yl-)benzamide65.N-tert-butyl-3-(2-(4-morpholinophenylamino)thieno[3,2-d]pyrimidin-7-yl)be-nzamide66.N-tert-butyl-3-(2-(4-(1-ethylpiperidin-4-yloxy)phenylamino)thieno[3,2-d]p-yrimidin-7-yl)benzenesulfonamide67. tert-butyl4-(2-(4-(morpholinomethyl)phenylamino)thieno[3,2-d]pyrimidin-7-yl)-1H-pyr-azole-1-carboxylate68.7-bromo-N-(4-((4-ethylpiperazin-1-yl)methyl)phenyl)thieno[3,2-d]pyrimidin-2-amine69.N-tert-butyl-3-(2-(4-((4-ethylpiperazin-1-yl)methyl)phenylamino)thieno[3,-2-d]pyrimidin-7-yl)benzenesulfonamide70.N-(4-((4-ethylpiperazin-1-yl)methyl)phenyl)-7-(1H-pyrazol-4-yl)thieno[3,2-d]pyrimidin-2-amine71.N-(cyanomethyl)-3-(2-(4-(morpholinomethyl)phenylamino)thieno[3,2-d]pyrimi-din-7-yl)benzamide72.N-tert-butyl-3-(2-(4-(2-(pyrrolidin-1-yl)ethoxy)phenylamino)thieno[3,2-d]-pyrimidin-7-yl)benzenesulfonamide73. tert-butylpyrrolidin-1-yl)ethoxy)phenylamino)thieno[3,2-d]pyrimidin-7-yl)benzylcarb-amate74.3-(2-(4-(2-(pyrrolidin-1-yl)ethoxy)phenylamino)thieno[3,2-d]pyri-midin-7-yl)benzenesulfonamide75.7-(3-chloro-4-fluorophenyl)-N-(4-(2-(pyrrolidin-1-yl)ethoxy)phenyl)thieno-[3,2-d]pyrimidin-2-amine76. tert-butyl4-(2-(4-(1-ethylpiperidin-4-yloxy)phenylamino)thieno[3,2-d]pyrimidin-7-yl-)-1H-pyrazole-1-carboxylate77.7-(benzo[d][1,3]dioxol-5-yl)-N-(4-(morpholinomethyl)phenyl)thieno[3,2-d]p-yrimidin-2-amine78. tert-butyl5-(2-(4-(morpholinomethyl)phenylamino)thieno[3,2-d]pyrimidin-7-yl)-1H-ind-ole-1-carboxylate79.7-(2-aminopyrimidin-5-yl)-N-(4-(morpholinomethyl)phenyl)thieno[3,2-d]pyri-midin-2-amine80. tert-butyl4-(2-(4-(morpholinomethyl)phenylamino)thieno[3,2-d]pyrimidin-7-yl)-5,6-di-hydropyridine-1(2H)-carboxylate81. tert-butylmorpholinomethyl)phenylamino)thieno[3,2-d]pyrimidin-7-yl)benzylcarbamate82.N-(3-(2-(4-(morpholinomethyl)phenylamino)thieno[3,2-d]pyrimidin-7-yl)-phenyl)acetamide83.N-(4-(2-(4-(morpholinomethyl)phenylamino)thieno[3,2-d]pyrimidin-7-yl)phen-yl)acetamide84.N-(3-(2-(4-(morpholinomethyl)phenylamino)thieno[3,2-d]pyrimidin-7-yl)phen-yl)methanesulfonamide85.7-(4-(4-methylpiperazin-1-yl)phenyl)-N-(4-(morpholinomethyl)phenyl)thieno-[3,2-d]pyrimidin-2-amine86.N-(2-methoxy-4-(2-(4-(morpholinomethyl)phenylamino)thieno[3,2-d]pyrimidin-7-yl)phenyl)acetamide87. 7-bromo-N-(3,4,5-trimethoxyphenyl)thieno[3,2-d]pyrimidin-2-amine 88.(3-(2-(3,4,5-trimethoxyphenylamino)thieno[3,2-d]pyrimidin-7-yl)phenyl)met-hanol89.(4-(2-(3,4,5-trimethoxyphenylamino)thieno[3,2-d]pyrimidin-7-yl)p-henyl)methanol90.(3-(2-(4-morpholinophenylamino)thieno[3,2-d]pyrimidin-7-yl)phenyl)methano-191.(4-(2-(4-morpholinophenylamino)thieno[3,2-d]pyrimidin-7-yl)phenyl)me-thanol92.N-(pyrrolidin-1-yl)ethoxy)phenylamino)thieno[3,2-d]pyrimidin-7-yl)benzyl)methanesulfonamide93. tert-butylmorpholinomethyl)phenylamino)thieno[3,2-d]pyrimidin-7-yl)benzylcarbamate94.N-(4-(morpholinomethyl)phenyl)-7-(3-(piperazin-1-yl)phenyl)thieno[3,2-d]pyrimidin-2-amine95.7-(6-(2-morpholinoethylamino)pyridin-3-yl)-N-(3,4,5-trimethoxyphenyl)thie-no[3,2-d]pyrimidin-2-amine96.7-(2-ethylphenyl)-N-(4-(2-(pyrrolidin-1-yl)ethoxy)phenyl)thieno[3,2-d]pyrimidin-2-amine97.7-(4-(aminomethyl)phenyl)-N-(4-(morpholinomethyl)phenyl)thieno[3,2-d]pyrimidin-2-amine98.N-(4-(1-ethylpiperidin-4-yloxy)phenyl)-7-(1H-pyrazol-4-yl)thieno[3,2-d]pyrimidin-2-amine99. N-(2,4-dimethoxyphenyl)-7-phenylthieno[3,2-d]pyrimidin-2-amine 100.7-bromo-N-(3,4-dimethoxyphenyl)thieno[3,2-d]pyrimidin-2-amine 101.N-(3,4-dimethoxyphenyl)-7-phenylthieno[3,2-d]pyrimidin-2-amine

R348 (Rigel) is defined in Velotta et al., “A novel JAK3 inhibitor,R348, attenuates chronic airway allograft rejection,” Transplantation.2009 Mar. 15; 87(5):653-9.

The present invention also relates to the pharmaceutically acceptableacid addition salts of compounds of Formulas A and B, as well as theadditional JAK inhibitors described herein. The acids which are used toprepare the pharmaceutically acceptable acid addition salts of theaforementioned base compounds of this invention are those which formnon-toxic acid addition salts, i.e., salts containing pharmacologicallyacceptable anions, such as the hydrochloride, hydrobromide, hydroiodide,nitrate, sulfate, bisulfate, phosphate, acid phosphate, acetate,lactate, citrate, acid citrate, tartrate, bitartrate, succinate,maleate, fumarate, gluconate, saccharate, benzoate, methanesulfonate,ethanesulfonate, benzenesulfonate, p-toluenesulfonate and pamoate [i.e.,1,1′-methylene-bis-(2-hydroxy-3-naphthoate)]salts.

The invention also relates to base addition salts of Formulas A and B.The chemical bases that may be used as reagents to preparepharmaceutically acceptable base salts of those compounds of Formulas Aand B that are acidic in nature are those that form non-toxic base saltswith such compounds. Such non-toxic base salts include, but are notlimited to those derived from such pharmacologically acceptable cationssuch as alkali metal cations (e.g., potassium and sodium) and alkalineearth metal cations (e.g., calcium and magnesium), ammonium orwater-soluble amine addition salts such asN-methylglucamine-(meglumine), and the lower alkanolammonium and otherbase salts of pharmaceutically acceptable organic amines.

The compounds of this invention include all conformational isomers(e.g., cis and trans isomers. The compounds of the present inventionhave asymmetric centers and therefore exist in different enantiomericand diastereomeric forms. This invention relates to the use of alloptical isomers and stereoisomers of the compounds of the presentinvention, and mixtures thereof, and to all pharmaceutical compositionsand methods of treatment that may employ or contain them. In thisregard, the invention includes both the E and Z configurations. Thecompounds of Formulas A and B can also exist as tautomers. Thisinvention relates to the use of all such tautomers and mixtures thereof.

This invention also encompasses pharmaceutical compositions containingprodrugs of compounds of the Formulas A and B, and their use in treatingor preventing HIV. This invention also encompasses methods of treatingor preventing viral infections that can be treated or prevented byprotein kinase inhibitors, such as the enzyme Janus Kinase 1, 2, or 3,comprising administering prodrugs of compounds of the Formulas A and B.Compounds of Formulas A and B having free amino, amido, hydroxy orcarboxylic groups can be converted into prodrugs. Prodrugs includecompounds wherein an amino acid residue, or a polypeptide chain of twoor more (e.g., two, three or four) amino acid residues which arecovalently joined through peptide bonds to free amino, hydroxy orcarboxylic acid groups of compounds of Formulas A and B. The amino acidresidues include the 20 naturally occurring amino acids commonlydesignated by three letter symbols and also include, 4-hydroxyproline,hydroxylysine, demosine, isodemosine, 3-methylhistidine, norvlin,beta-alanine, gamma-aminobutyric acid, citrulline, homocysteine,homoserine, ornithine and methioine sulfone. Prodrugs also includecompounds wherein carbonates, carbamates, amides and alkyl esters whichare covalently bonded to the above substituents of Formulas A and Bthrough the carbonyl carbon prodrug sidechain.

II. Combinations of JAK Inhibitors and Other Antiviral Agents

In one embodiment, the compositions include antiretroviral JAKinhibitors as described herein and one or more additional antiviralagents.

In one aspect of this embodiment, the JAK inhibitors and additionalantiviral agents are administered in combination or alternation, and inone aspect, in a manner in which both agents act synergistically againstthe virus. The compositions and methods described herein can be used totreat patients infected with a drug resistant form of HIV, specifically,a form including the M184V/I, multidrug resistant viruses (e.g., Q151M),K65R mutation, Thymidine analog mutations (TAMS), and the like. TAMSinclude, but are not limited to, mutations at reverse transcriptase (RT)positions 41, 67, 70, 210, 215, and 219, which confer clinicallysignificant resistance to each of the nucleoside RT inhibitors with theexception of 3TC.

While not wishing to be bound to a particular theory, it is believedthat the JAK inhibitors described herein function in a way notassociated with heretofore known antiretroviral therapy, in that thecompounds do not act in the same way as NRTI, NNRTI, proteaseinhibitors, integrase inhibitors, entry inhibitors, and the like, all ofwhich interfere directly with a step in the viral replication cycle.Rather, they act in an intracellular manner, in a way that is not likelyto provoke resistance. More specifically, the mechanism is independentand distinct from direct modulation or interference with the viralreplication cycle itself, and therefore lacks a selective pressure toconfer emergence of drug resistant virus.

Further, the combination of the JAK inhibitors described herein, and oneor more additional antiviral agents, can help prevent the development ofviral resistance to other antiviral agents. Therefore, co-formulation ofthe JAK inhibitors with these additional antiviral agents can functionas a “resistance repellent” for the various mutations associated withconventional therapy, and provides better therapy than either alone.

In one aspect of this embodiment, a combination therapy is administeredthat has the capability of attacking HIV in a variety of mechanisms.That is, the combination therapy includes an effective amount of atleast one adenine, cytosine, thymine, and guanosine nucleosideantiviral, as well as one or more additional agents other than NRTI thatinhibit HIV viral loads via a different mechanism. Examples includereverse transcriptase inhibitors, protease inhibitors, fusioninhibitors, entry inhibitors, attachment inhibitors, polymeraseinhibitors, and integrase inhibitors such as integrase inhibitors suchas raltegravir (Isentress) or MK-0518, GS-9137 (Gilead Sciences),GS-8374 (Gilead Sciences), or GSK-364735.

It is believed that this therapy, particularly when administered at anearly stage in the development of HIV infection, has the possibility ofeliminating HIV infection in a patient. That is, the presence of thedifferent nucleosides and additional agents minimizes the ability of thevirus to adapt its reverse transcriptase and develop resistance to anyclass of nucleoside antiviral nucleosides (i.e., adenine, cytosine,thymidine, or guanine), because it would be susceptible to at least oneof the other nucleoside antiviral agents that are present, and/or theadditional non-NRTI therapeutic agent. In addition the lipophiliccharacter of certain agents would allow them to penetrate certaincompartments where virus could replicate (e.g., brain, testicles, gut).

Representative agents are described in more detail below.

Attachment and Fusion Inhibitors

Attachment and fusion inhibitors are anti-HIV drugs which are intendedto protect cells from infection by HTV by preventing the virus fromattaching to a new cell and breaking through the cell membrane. Thesedrugs can prevent infection of a cell by either free virus (in theblood) or by contact with an infected cell. These agents are susceptibleto digestive acids, so are commonly delivered by break them down, mostof these drugs are given by injections or intravenous infusion.

Examples are shown in the table that follows:

Entry Inhibitors (including Fusion Inhibitors) Brand GenericExperimental Pharmaceutical Name Name Abbreviation Code Company Fuzeon ™enfuvirtide T-20 Trimeris T-1249 Trimeris AMD-3100 AnorMED, Inc.CD4-IgG2 PRO-542 Progenics Pharmaccuticals BMS-488043 Bristol-MycrsSquibb aplaviroc GSK-873,140 GlaxoSmithKline Peptide T Advanced ImmuniT, Inc. TNX-355 Tanox, Inc. maraviroc UK-427,857 Pfizer CXCR4 InhibitorAMD070 AMD11070 AnorMED, Inc. CCR5 antagonist Vicriroc SCH-D SCH-417690Schering-Plough

Additional fusion and attachment inhibitors in human trials includeAK602, AMD07, BMS-378806, HGS004, NC9471, PRO 140, Schering C, SP01A,and TAK-652.

AK602 is a CCR5 blocker being developed by Kumamoto University in Japan.

AMD070 by AnorMed blocks the CXCR4 receptor on CD4 T-cells to inhibitHIV fusion.

BMS-378806 is an attachment inhibitor that attaches to gp120, apart ofHIV.

HGS004 by Human Genome Sciences, is a monoclonal antibody CCR5 blocker.

INCB 9471 is sold by Incyte Corporation.

PRO 140 by Progenics blocks fusion by binding to a receptor protein onthe surface of CD4 cells.

SP01A by Samaritan Pharmaceuticals is an HIV entry inhibitor.

TAK-652 by Takeda blocks binding to the CCR5 receptor.

Polymerase Inhibitors

The DNA polymerization activity of HTV-1 reverse transcriptase (RT) canbe inhibited by at least three mechanistically distinct classes ofcompounds. Two of these are chain terminating nucleoside analogs (NRTIs)and allosteric non-nucleoside RT inhibitors (NNRTIs). The third classincludes pyrophosphate mimetics such as foscarnet (phosphonoformic acid,PFA).

The reverse transcriptase has a second enzymatic activity, ribonucleaseH (RNase H) activity, which maps to a second active site in the enzyme.RNase H activity can be inhibited by various small molecules (polymeraseinhibitors). Examples include diketo acids, which bind directly to theRNase H domain, or compounds like PFA, which are believed to bind in thepolymerase domain.

Examples of these compounds are listed in the tables that follow.

HIV Therapies: Nucleoside/Nucleotide Reverse

Transcriptase Inhibitors (NRTIs) Brand Generic ExperimentalPharmaceutical Name Name Abbreviation Code Company Dapavir, 2,6- DAPDRFS Pharma diaminopurine dioxolane Retrovir ® zidovudine AZT or ZDVGlaxoSmithKline Epivir ® lamivudinc 3TC GlaxoSmithKlinc Combivir ®zidovudine + AZT + 3TC GlaxoSmithKline lamivudine Trizivir ® abacavir +ABC + AZT + GlaxoSmithKline zidovudine + 3TC lamivudine Ziagen ®abacavir ABC 1592U89 GlaxoSmithKline Epzicom ™ abacavir + ABC + 3TCGlaxoSmithKline lamivudine Hivid ® zalcitabine ddC Hoffmann-La RocheVidex ® didanosine: ddI BMY-40900 Bristol-Myers buffered Squibb versionsEntecavir baraclude Bristol-Myers Squibb Videx ® EC didanosine: ddIBristol-Myers delayed- Squibb release capsulcs Zcrit ® stavudinc d4TBMY-27857 Bristol-Mvers Squibb Viread ™ tcnofovir TDF or Gilead Sciencesdisoproxil Bis(POC) fumarate (DF) PMPA Emtriva ® emtricitabine (−)-FTCGilead Sciences Truvada ® Vircad + TDF + (−)- Gilead Sciences EmtrivaFTC Atripla ™ TDF + (−)- Gilead/BMS/Merck FTC + Sustiva ® AmdoxovirDAPD, RFS Pharma EEC AMDX Apricitabine AVX754 SPD 754 Avexa LtdAlovudine FLT MIV-310 Medivir Elvucitabine L-FD4C ACH-126443, AchillionKP-1461 SN1461, Koronis SN1212 Racivir RCV Emory University DOT EmoryUniversity Dexelvucitabine Reverset D-D4FC, DFC DPC 817 Emory UniversityGS9148 and Gilead Sciences prodrugs thereof

HIV Therapies: Non-Nucleoside Reverse

Transcriptase Inhibitors (NNRTIs) Brand Generic ExperimentalPharmaceutical Name Name Abbreviation Code Company Viramune ® nevirapineNVP BI-RG-587 Boehringer Ingelheim Rescriptor ® delavirdine DLVU-90152S/T Pfizer Sustiva ® efavirenz EFV DMP-266 Bristol-Myers Squibb(+)-calanolide A Sarawak Medichem capravirine CPV AG-1549 or PfizerS-1153 DPC-083 Bristol-Myers Squibb TMC-125 Tibotec-Virco Group TMC-278Tibotec-Virco Group IDX12899 Idenix IDX12989 Idenix RDEA806 ArdeaBioscience, Inc.

Integrase Inhibitors

Representative integrase inhibitors include globoidnan A, L-000870812,S/GSK1349572, S/GSK1265744, Raltegravir and Elvitegravir with or withouta pharmacokinetic (PK) booster such as ritonavir or Gilead'spharmacoenhancing agent (also referred to as a PK booster), GS 9350.

Suitable integrase inhibitors include those described in:

U.S. patent application Ser. No. 11/595,429, entitled “HIV INTEGRASEINHIBITORS” filed in the name of B. Narasimhulu Naidu, et al. on Nov.10, 2006 and published on May 17, 2007 as U.S. Publication No.20070111985 and assigned to Bristol-Meyers Squibb Company.

U.S. patent application Ser. No. 11/561,039, entitled “HIV INTEGRASEINHIBITORS” filed in the name of B. Narasimhulu Naidu, et al. on Nov.17, 2006 and published on Jun. 7, 2007 as U.S. Publication No.20070129379 and assigned to Bristol-Meyers Squibb Company.

U.S. patent application Ser. No. 11/599,580, entitled “HIV INTEGRASEINHIBITORS” filed in the name of B. Narasimhulu Naidu, et al. on Nov.14, 2006 and published on May 17, 2007 as U.S. Publication No.20070112190 and assigned to Bristol-Meyers Squibb Company.

U.S. patent application Ser. No. 11/754,462, entitled “HIV INTEGRASEINHIBITORS” filed in the name of B. Narasimhulu Naidu, et al. on May 29,2007 and published on Dec. 6, 2007 as U.S. Publication No. 20070281917and assigned to Bristol-Meyers Squibb Company.

U.S. patent application Ser. No. 11/768,458, entitled “HIV INTEGRASEINHIBITORS” filed in the name of Michael A. Walker, et al. on Jun. 26,2007 and published Jan. 3, 2008 as U.S. Publication No. 20080004265 andassigned to Bristol-Meyers Squibb Company.

U.S. patent application Ser. No. 12/132,145, entitled “HIV INTEGRASEINHIBITORS” filed in the name of B. Narasimhulu Naidu, et al. on Jun. 3,2008; published on Dec. 11, 2008 as U.S. Publication No. 20080306051 andassigned to Bristol-Meyers Squibb Company.

U.S. patent application Ser. No. 11/505,149, entitled “BICYCLICHETEROCYCLES AS HIV INTEGRASE INHIBITORS” filed in the name of B.Narasimhulu Naidu, et al. on Aug. 16, 2006 and published on Dec. 7, 2006as U.S. Publication No. 20060276466.

U.S. patent application Ser. No. 11/590,637, entitled “HIV INTEGRASEINHIBITORS” filed in the name of B. Narasimhulu Naidu, et al. on Oct.31, 2006 and published on May 17, 2007 as U.S. Publication No.20070111984 and assigned to Bristol-Meyers Squibb Company.

U.S. patent application Ser. No. 12/162,975, entitled “USE OF6-(3-CHLORO-2-FLUOROBENZYL)-1-[(2S)-1-HYDROXY-3-METHYLBUTAN-2-YL]-7-METHOXY-4-OXO-1,4-DIHYDROQUINOLINE-3-CARBOXYLICACID OR SALT THEREOF FOR TREATING RETROVIRUS INFECTION” filed in thename of Yuji Matsuzaki, et al. on Feb. 1, 2007 and published on Jan. 15,2009 as U.S. Publication No. 20090018162.

U.S. patent application Ser. No. 11/767,021, entitled“6-(HETEROCYCLYL-SUBSTITUTED BENZYL)-4-OXOQUINOLINE COMPOUND AND USETHEREOF AS HIV INTEGRASE INHIBITOR” filed in the name of Motohid, Satoh,et al. on Jun. 22, 2007 and published on Aug. 28, 2008 as U.S.Publication No. 20080207618.

U.S. patent application Ser. No. 12/042,628, entitled “USE OF QUINOLINEDERIVATIVES WITH ANTI-INTEGRASE EFFECT AND APPLICATIONS THEREOF” filedin the name of Aurelia Mousnier, et al. on Mar. 5, 2008 and published onJul. 3, 2008 as U.S. Publication No. 20080161350 and assigned toBioalliance Pharma SA.

U.S. patent application Ser. No. 12/169,367, entitled “NOVELPYRIMIDINECARBOXAMIDE DERIVATIVES” filed in the name of Scott L.Harbeson on Jul. 8, 2008 and published on Feb. 5, 2009 as U.S.Publication No. 20090035324.

U.S. patent application Ser. No. 10/587,857, entitled “NAPHTHYRIDINEDERIVATIVES HAVING INHIBITORY ACTIVITY AGAINST HIV INTEGRASE” filed inthe name of Teruhiko Taishi, et al. on Feb. 2, 2005 and published onSep. 10, 2009 as U.S. Publication No. 20090227621.

U.S. patent application Ser. No. 11/500,387, entitled“NITROGEN-CONTAINING HETEROARYL COMPOUNDS HAVING INHIBITORY ACTIVITYAGAINST HIV INTEGRASE” filed in the name of Masahiro Fuji, et al. onAug. 8, 2006 and published on Dec. 28, 2006 as U.S. Publication No.20060293334.

U.S. patent application Ser. No. 12/097,859, entitled “METHODS FORIMPROVING THE PHARMACOKINETICS OF HIV INTEGRASE INHIBITORS” filed in thename of Brian P. Kearney, et al. on Dec. 29, 2006 and published on Sep.17, 2009 as U.S. Publication No. 20090233964 and assigned to GileadSciences, Inc.

U.S. patent application Ser. No. 11/807,303, entitled “PRE-ORGANIZEDTRICYCLIC INTEGRASE INHIBITOR COMPOUNDS” filed in the name of James M.Chen, et al. on May 25, 2007 and published on Jan. 29, 2009 as U.S.Publication No. 20090029939 and assigned to Gilead Sciences, Inc.

U.S. patent application Ser. No. 10/587,601, entitled “HIV INTEGRASEINHIBITORS” filed in the name of Philip Jones, et al. on Mar. 1, 2005and published on Jul. 12, 2007 as U.S. Publication No. 20070161639 andassigned to Merck and Co., Inc.

U.S. patent application Ser. No. 10/592,222, entitled “HIV INTEGRASEINHIBITORS” filed in the name of Peter D. Jones, et al. on Mar. 4, 2005and published on Jan. 10, 2008 as U.S. Publication No. 20080009490 andassigned to Merck and Co., Inc.

U.S. patent application Ser. No. 11/992,531, entitled “HIV INTEGRASEINHIBITORS” filed in the name of Vincenzo Summa, et al. on Sep. 26, 2006and published on Sep. 3, 2009 as U.S. Publication No. 20090221571 andassigned to Merck and Co., Inc.

U.S. patent application Ser. No. 10/587,682, entitled “HIV INTEGRASEINHIBITORS” filed in the name of Wei Han, et al. on Mar. 9, 2005 andpublished on Aug. 2, 2007 as U.S. Publication No. 20070179196 andassigned to Merck and Co., Inc.

U.S. patent application Ser. No. 11/641,508, entitled “N-SUBSTITUTEDHYDROXYPYRIMIDINONE CARBOXAMIDE INHIBITORS OF HIV INTEGRASE” filed inthe name of Benedetta Crescenzi, et al. on Dec. 19, 2006 and publishedon May 31, 2007 as U.S. Publication No. 20070123524 and assigned toMerck and Co., Inc.

U.S. patent application Ser. No. 11/435,671, entitled “INTEGRASEINHIBITOR COMPOUNDS” filed in the name of Zhenhong R. Cai, et al. on May16, 2006 and published on Mar. 29, 2007 as U.S. Publication No.20070072831 and assigned to Gilead Sciences, Inc.

U.S. patent application Ser. No. 11/804,041, entitled “INTEGRASEINHIBITORS” filed in the name of Zhenhong R. Cai, et al. on May 16, 2007and published on Mar. 6, 2008 as U.S. Publication No. 20080058315 andassigned to Gilead Sciences, Inc.

U.S. patent application Ser. No. 11/880,854, entitled “NOVEL HIV REVERSETRANSCRIPTASE INHIBITORS” filed in the name of Hongyan Guo, et al. onJul. 24, 2007 and published on Mar. 20, 2008 as U.S. Publication No.20080070920 and assigned to Gilead Sciences, Inc.

U.S. patent application Ser. No. 10/585,504, entitled “PYRIMIDYLPHOSPHONATE ANTIVIRAL COMPOUNDS AND METHODS OF USE” filed in the name ofHaolun Jin, et al. on Nov. 1, 2005 and published on Jun. 26, 2008 asU.S. Publication No. 20080153783 and assigned to Gilead Sciences, Inc.

U.S. patent application Ser. No. 11/579,772, entitled “HIV INTEGRASEINHIBITORS” filed in the name of John S. Wai, et al. on May 3, 2005 andpublished on Nov. 20, 2008 as U.S. Publication No. 20080287394 andassigned to Merck and Co., Inc.

U.S. patent application Ser. No. 10/591,914, entitled “HIV INTEGRASEINHIBITORS” filed in the name of Matthew M. Morrissette, et al. on Mar.4, 2005 and published on Jun. 12, 2008 as U.S. Publication No.20080139579 and assigned to Merck and Co., Inc.

U.S. patent application Ser. No. 11/629,153, entitled “HTV INTEGRASEINHIBITORS” filed in the name of John S. Wai, et al. on Jun. 3, 2005 andpublished on Jun. 18, 2008 as U.S. Publication No. 20080015187 andassigned to Merck and Co., Inc.

U.S. patent application Ser. No. 12/043,636, entitled “HIV INTEGRASEINHIBITORS, PHARMACEUTICAL COMPOSITIONS AND METHOD FOR THEIR USE” filedin the name of Qiyue Hu, et al. on Mar. 6, 2008 and published on Sep.11, 2008 as U.S. Publication No. 20080221154 and assigned to Pfizer,Inc.

PCT WO 2007/019098, entitled “HIV INTEGRASE INHIBITORS,” listingSmithKline Beecham Corporation, Shionogi & Co. Ltd., and TakashiKawasuji as applicants, and Brian Johns as an inventor, published onFeb. 15, 2007.

U.S. patent application Ser. No. 12/306,198, entitled “MODULATORS OFPHARMACOKINETIC PROPERTIES OF THERAPEUTICS” filed in the name of Desai,Manoj C., et al. and was published on Nov. 26, 2009 as U.S. PublicationNo. 20090291952 and is assigned to Gilead Sciences, Inc.

U.S. patent application Ser. No. 12/274,107, entitled, “INTEGRASEINHIBITORS” filed Nov. 19, 2008 in the name of Jabri, Salman Y., et al.and was published on Nov. 26, 2009 as U.S. Publication No. 20090291921and is assigned to Gilead Sciences, Inc.

U.S. patent application Ser. No. 12/215,605 “ANTIVIRAL COMPOUNDS” filedon Jun. 26, 2008 in the name of Cho, Aesop, et al., and was published onOct. 15, 2009 as U.S. Publication No. 20090257978 and is assigned toGilead Sciences, Inc.

U.S. patent application Ser. No. 12/097,859 METHODS FOR IMPROVING THEPHARMACOKINETICS OF HIV INTEGRASE INHIBITORS filed on Dec. 29, 2006 inthe name of Kearney; Brian P., et al. and published on Sep. 17, 2009 asU.S. Publication No. 20090233964 and assigned to Gilead Sciences, Inc.

U.S. patent application Ser. No. 11/658,419, entitled “PHOSPHONATEANALOGS OF HIV INHIBITOR COMPOUNDS” filed Jul. 26, 2005 in the name ofBoojamra; Constantine G., et al. and was published on Aug. 13, 2009 asU.S. Publication No. 20090202470 and is assigned to Gilead Sciences,Inc.

U.S. patent application Ser. No. 12/215,601, entitled, “ANTIVIRALCOMPOUNDS” filed on Jun. 26, 2008 in the name of Cottell, Jeromy J., etal. and published on Jul. 23, 2009 as U.S. Publication No. 20090186869and assigned to Gilead Sciences, Inc.

U.S. patent application Ser. No. 12/217,496 entitled “MODULATORS OFPHARMACOKINETIC PROPERTIES OF THERAPEUTICS” in the name of Desai, ManojC., et al. and published on Jul. 16, 2009 as U.S. Publication No.20090181902 and assigned to Gilead Sciences, Inc.

U.S. patent application Ser. No. 12/340,419 entitled “INHIBITORS OFCYTOCHROME P450” filed on Dec. 19, 2008 in the name of Desai, Manoj C.et al. and published on Jul. 9, 2009 as U.S. Publication No. 20090175820and assigned to Gilead Sciences, Inc.

U.S. patent application Ser. No. 12/195,161 entitled “COMPOSITIONS ANDMETHODS FOR COMBINATION ANTIVIRAL THERAPY” filed on Aug. 20, 2008 in thename of Dahl, Terrence C. et al. and published on Jun. 4, 2009 as U.S.Publication No. 20090143314 and assigned to Gilead Sciences, Inc.

U.S. patent application Ser. No. 12/208,952 entitled “PROCESS ANDINTERMEDIATES FOR PREPARING INTEGRASE INHIBITORS” filed on Sep. 11, 2008in the name of Dowdy, Eric, et al. and published on Apr. 16, 2009 asU.S. Publication No. 20090099366 and assigned to Gilead Sciences, Inc.

U.S. patent application Ser. No. 12/147,220 entitled “THERAPEUTICCOMPOSITIONS AND METHODS” filed on Jun. 26, 2008 in the name of Kearney,Brian P. et al and published on Apr. 9, 2009 as U.S. Publication No.20090093482 and assigned to Gilead Sciences, Inc.

U.S. patent application Ser. No. 12/147,041 entitled “THERAPEUTICCOMPOSITIONS AND METHODS” filed on Jun. 26, 2008 in the name of Kearney,Brian P. et al., published on Apr. 9, 2009 as U.S. Publication No.20090093467 and assigned to Gilead Sciences, Inc.

U.S. patent application Ser. No. 12/215,266 entitled “ANTIVIRALCOMPOUNDS” filed on Jun. 26, 2008 in the name of Cai, Zhenhong R. etal., published Feb. 19, 2009 as U.S. Publication No. 20090047252 andassigned to Gilead Sciences, Inc.

U.S. patent application Ser. No. 12/204,174 entitled “COMPOSITIONS ANDMETHODS FOR COMBINATION ANTIVIRAL THERAPY” filed on Sep. 4, 2008 in thename of Dahl, Terrence C., et al., published on Feb. 5, 2009 as U.S.Publication No. 20090036408 and assigned to Gilead Sciences, Inc.

U.S. patent application Ser. No. 10/585,504 entitled “PYRIMIDYLPHOSPHONATE ANTIVIRAL COMPOUNDS AND METHODS OF USE” filed on Nov. 1,2005 in the name of Jin, Haolun et al., published on Jun. 26, 2008 asU.S. Publication No. 20080153783 and assigned to Gilead Sciences, Inc.

U.S. patent application Ser. No. 11/853,606 entitled “PROCESS ANDINTERMEDIATES FOR PREPARING INTEGRASE INHIBITORS” filed on Sep. 11, 2007in the name of Dowdy, Eric, et al, published May 29, 2008 as U.S.Publication No. 20080125594 and assigned to Gilead Sciences, Inc.

U.S. patent application Ser. No. 11/644,811 entitled “PROCESSES ANDINTERMEDIATES USEFUL FOR PREPARING INTEGRASE INHIBITOR COMPOUNDS” filedon Dec. 21, 2006 in the name of Evans, Jared W. et al., published onFeb. 14, 2008 as U.S. Publication No. 20080039487 and assigned to GileadSciences, Inc.

U.S. patent application Ser. No. 10/586,627 entitled “USE OF ADEFOVIR ORTENOFOVIR FOR INHIBITING MMTV-LIKE VIRUSES INVOLVED IN BREAST CANCER ANDPRIMARY BILIARY CIRRHOSIS” filed on Jul. 20, 2007 in the name of Cihlar,Tomas, et al., published on Dec. 6, 2007 as U.S. Publication No.20070281911 and assigned to Gilead Sciences, Inc.

U.S. patent application Ser. No. 11/435,671 entitled “INTEGRASEINHIBITOR COMPOUNDS” filed on May 16, 2006 in the name of Cai, ZhenhongR. et al., published on Mar. 29, 2007 as U.S. Publication No.20070072831 and assigned to Gilead Sciences, Inc.

U.S. patent application Ser. No. 11/190,225 entitled “PHOSPHONATEANALOGS OF HIV INHIBITOR COMPOUNDS” filed on Jul. 26, 2005 in the nameof Boojamra, Constantine G. et al., published on Mar. 1, 2007 as U.S.Publication No. 20070049754 and assigned to Gilead Sciences, Inc.

U.S. patent application Ser. No. 10/511,182 entitled “NON NUCLEOSIDEREVERSE TRANSCRIPTASE INHIBITORS” filed on Feb. 28, 2005 in the name ofChen, James M. et al., published on Jun. 15, 2006 as U.S. PublicationNo. 20060128692 and assigned to Gilead Sciences, Inc.

U.S. patent application Ser. No. 11/033,422 entitled “PYRIMIDYLPHOSPHONATE ANTIVIRAL COMPOUNDS AND METHODS OF USE” filed on Jan. 11,2005 in the name of Jin, Haolun et al., published on Dec. 22, 2005 asU.S. Publication No. 20050282839 and assigned to Gilead Sciences, Inc.

U.S. patent application Ser. No. 11/040,929 entitled “METHODS OFINHIBITION OF MMTV-LIKE VIRUSES” filed on Jan. 21, 2005 in the name ofCihlar, Tomas et al., published on Oct. 27, 2005 as U.S. Publication No.20050239753 and assigned to Gilead Sciences, Inc.

U.S. patent application Ser. No. 10/423,496 entitled “CELLULARACCUMULATION OF PHOSPHONATE ANALOGS OF HIV PROTEASE INHIBITOR COMPOUNDS”filed on Apr. 25, 2003 in the name of Arimilli, Murty N. et al.,published on Sep. 22, 2005 as U.S. Publication No. 20050209197 andassigned to Gilead Sciences, Inc.

U.S. patent application Ser. No. 10/424,130 entitled “NON NUCLEOSIDEREVERSE TRANSCRIPTASE INHIBITORS” filed on Apr. 25, 2003 in the name ofChen, James M. et al., published on Sep. 8, 2005 as U.S. Publication No.20050197320 and assigned to Gilead Sciences, Inc.

U.S. patent application Ser. No. 10/944,118 entitled “AZA-QUINOLINOLPHOSPHONATE INTEGRASE INHIBITOR COMPOUNDS” filed on Sep. 17, 2004 in thename of Jin, Haolun et al., published on Jun. 23, 2005 as U.S.Publication No. 20050137199 and assigned to Gilead Sciences, Inc.

U.S. patent application Ser. No. 10/903,288 entitled “NUCLEOBASEPHOSPHONATE ANALOGS FOR ANTIVIRAL TREATMENT” filed on Jul. 30, 2004 inthe name of Krawezyk, Steven H., published on Mar. 17, 2005 as U.S.Publication No. 20050059637 and assigned to Gilead Sciences, Inc.

U.S. patent application Ser. No. 10/757,141 entitled “COMPOSITIONS ANDMETHODS FOR COMBINATION ANTIVIRAL THERAPY” filed Jan. 13, 2004 Dahl,Terrance C. et al., published on Nov. 11, 2004 as U.S. Publication No.20040224917 and assigned to Gilead Sciences, Inc.

U.S. patent application Ser. No. 10/757,122 entitled “COMPOSITIONS ANDMETHODS FOR COMBINATION ANTIVIRAL THERAPY” filed on Jan. 13, 2004 Dahl,Terrance C. et al., published on Nov. 11, 2004 as U.S. Publication No.20040224916 and assigned to Gilead Sciences, Inc.

U.S. patent application Ser. No. 10/687,373 entitled “PRE-ORGANIZEDTRICYCLIC INTEGRASE INHIBITOR COMPOUNDS” filed on Oct. 16, 2003 in thename of Chen, James M. et al., published on Aug. 26, 2004 as U.S.Publication No. 20040167124 and assigned to Gilead Sciences, Inc.

U.S. patent application Ser. No. 10/687,374 entitled “PRE-ORGANIZEDTRICYCLIC INTEGRASE INHIBITOR COMPOUNDS” filed on Oct. 15, 2003 in thename of Chen, James M. et al., published on Aug. 12, 2004 as U.S.Publication No. 20040157804 and assigned to Gilead Sciences, Inc.

U.S. patent application Ser. No. 10/424,186 entitled “METHOD ANDCOMPOSITIONS FOR IDENTIFYING ANTI-HIV THERAPEUTIC COMPOUNDS” filed onApr. 25, 2003 in the name of Birkus, Gabriel et al., published on Jun.24, 2004 as U.S. Publication No. 20040121316 and assigned to GileadSciences, Inc.

U.S. patent application Ser. No. 11/820,444 entitled “DIKETO ACIDS WITHNUCLEOBASE SCAFFOLDS: ANTI-HIV REPLICATION INHIBITORS TARGETED AT HIVINTEGRASE” filed on Jun. 19, 2007 in the name of Nair, Vasu et al.,published on Nov. 8, 2007 as U.S. Publication No. 20070259823 andassigned to the University of Georgia Research Foundation, Inc.

U.S. patent application Ser. No. 11/047,229 entitled “DIKETO ACIDS WITHNUCLEOBASE SCAFFOLDS: ANTI-HIV REPLICATION INHIBITORS TARGETED AT HIVINTEGRASE” filed on Jan. 31, 2005 in the name of Nair, Vasu et al.,published on Aug. 3, 2006 as U.S. Publication No. 20060172973.

U.S. patent application Ser. No. 11/827,959 entitled “PYRIDINONE DIKETOACIDS: INHIBITORS OF HIV REPLICATION” filed on Jul. 13, 2007 in the nameof Nair, Vasu et al., published on Jan. 24, 2008 as U.S. Publication No.20080020010 and assigned to the University of Georgia ResearchFoundation, Inc.

Additional integrase inhibitors include L-870, 810 (Merck), INH-001(Inhibitex), L870810 (Merck), PL-2500, composed of pryidoxal1-5-phosphate dcrivativcs (Procyon) monophores (Suncsis), V-165 (RegaInstitute, Belgium), Mycelium integrasone (a fungal polyketide, Merck),GS 9224 (Gilead Sciences), AVX-I (Avexa), ITI-367, an oxadiazolpre-integrase inhibitor (George Washington University), GSK364735(GSK/Shionogi), GS-9160 (GSK), S-1360 (Shionogi-GlaxoSmithKlinePharmaceuticals LLC), RSC 1838 (GSK/Shionogi), GS-9137 (taken alone orwith Norvir) (Gilead), MK-2048 (Merck), S/GSK 1349572 and S/GSK 1265744(no need for a PK booster) (GSK/Shionogi),6-(3-chloro-2-fluorobenzyl)-1-[(2S)-1-hydroxy-3-methylbutan-2-y-l]-7-methoxy-4-oxo-1,4-dihydroquinoline-3-carboxylicacid (U.S. Patent Application Publication No. 20090018162), 5-1360,L-870810, MK-0518 (Merck), C-2507 (Merck), BMS 538158 (Bristol MyersSquibb), and L-900564 (Merck).

The structure of L-900564 is shown below:

Nair et al., J Med Chem. 2006 Jan. 26; 49(2): 445-447, discloses thefollowing integrase inhibitors:

Additional integrase inhibitors are disclosed in Pais et al., J MedChem. 2002 Jul. 18; 45(15):3184-94.

Several integrase inhibitors are peptides, including those disclosed inDivita et al., Antiviral Research, Volume 71, Issues 2-3, September2006, Pages 260-267.

Another integrase inhibitor that can be used in the methods of treatmentdescribed herein include 118-D-24, which is disclosed, for example, inVatakis, Journal of Virology, April 2009, p. 3374-3378, Vol. 83, No. 7.

Additional integrase inhibitors include those described in McKeel etal., “Dynamic Modulation of HIV-1 Integrase Structure and Function byCellular LEDGF Protein, JBC Papers in Press. Published on Sep. 18, 2008as Manuscript M805843200.

Other representative integrase inhibitors include dicaffeoylquinic acids(DCQAs), such as those disclosed in Zhu et al., “Irreversible Inhibitionof Human Immunodeficiency Virus Type 1 Integrase by DicaffeoylquinicAcids,” Journal of Virology, April 1999, p. 3309-3316, Vol. 73, No. 4.

There are also various nucleoside compounds active as integraseinhibitors, including those disclosed in Mazumder, A., N. Neamati, J. P.Sommadossi, G. Gosselin, R. F. Schinazi, J. L. Imbach, and Y. Pommier.1996. Effects of nucleotide analogues on human immunodeficiency virustype 1 integrase. Mol. Pharmacol. 49:621-628.

Protease Inhibitors

Protease inhibitors treat or prevent HIV infection by preventing viralreplication. They act by inhibiting the activity of HIV protease, anenzyme that cleaves nascent proteins for final assembly of new virons.Examples are shown in the table that follows.

HIV Therapies: Protease Inhibitors (PIs) Brand Generic ExperimentalPharmaceutical Name Name Abbreviation Code Company Invirase ® saquinavir(Hard SQV (HGC) Ro-31-8959 Hoffmann-La Roche Gel Cap) Fortovase ®saquinavir (Soft SQV (SGC) Hoffmann-La Roche Gel Cap) Norvir ® RitonavirRTV ABT-538 Abbott Laboratories Crixivan ® Indinavir IDV MK-639 Merck &Co. Viracept ® Nelfinavir NFV AG-1343 Pfizer Agenerase ® Amprenavir APV141W94 or Glaxo SmithKline VX-478 Kaletra ® lopinavir + LPV ABT-378/rAbbott Laboratories ritonavir Lexiva ® fosamprenavir GW-433908 or GlaxoSmithKline VX-175 Aptivus ® tripanavir TPV PNU-140690 BoehringerIngelheim Reyataz ® atazanavir BMS-232632 Bristol-Myers SquibbBrecanavir GW640385 Glaxo SmithKline Prczista ™ Darunavir TMC114 Tibotec

HIV Therapies: Other Classes of Drugs Brand Generic ExperimentalPharmaceutical Name Name Abbreviation Code Company Viread ™ tenofovirTDF or Gilead Sciences disoproxil Bis(POC) fumarate PMPA (DF)

Cellular Inhibitors Brand Generic Experimental Pharmaceutical Name NameAbbreviation Code Company Droxia ® Hydroxyurea HU Bristol-Myers Squibb

HIV Therapies: Immune-Based Therapies Brand Generic ExperimentalPharmaceutical Name Name Abbreviation Code Company Proleukin ®aldesleukin, or IL-2 Chiron Corporation Interleukin-2 Remune ® HIV-1AG1661 The Immune Immunogen, or Response Corporation Salk vaccine HE2000HollisEden Pharmaceuticals

III. Combination or Alternation HIV-Agents

In general, during alternation therapy, an effective dosage of eachagent is administered serially, whereas in combination therapy, aneffective dosage of two or more agents is administered together. Inalternation therapy, for example, one or more first agents can beadministered in an effective amount for an effective time period totreat the viral infection, and then one or more second agentssubstituted for those first agents in the therapy routine and likewisegiven in an effective amount for an effective time period.

The dosages will depend on such factors as absorption, biodistribution,metabolism and excretion rates for each drug as well as other factorsknown to those of skill in the art. It is to be noted that dosage valueswill also vary with the severity of the condition to be alleviated. Itis to be further understood that for any particular subject, specificdosage regimens and schedules should be adjusted over time according tothe individual need and the professional judgment of the personadministering or supervising the administration of the compositions.

Examples of suitable dosage ranges for anti-HIV compounds, including theJAK inhibitors described herein, can be found in the scientificliterature and in the Physicians Desk Reference. Many examples ofsuitable dosage ranges for other compounds described herein are alsofound in public literature or can be identified using known procedures.These dosage ranges can be modified as desired to achieve a desiredresult.

Certain JAK inhibitors described herein are also inhibitors of CYP3A4,which means that they will significantly increase the C_(max) plasmalevel of any anti-HIV drug that binds to CYP3A4, including HIV-1protease inhibitors. This information can be taken into considerationwhen determining suitable dosages for such compounds.

IV. Combination Therapy for Treating an HCV Infection

Nonlimiting examples of additional agents include:

HCV Protease inhibitors: Examples include Medivir HCV Protease Inhibitor(HCV-PI or TMC435) (Medivir/Tibotec); MK-7009 (Merck), RG7227 (ITMN-191)(Roche/Pharmasset/InterMune), boceprevir (SCH 503034) (Schering), SCH446211 (Schering), narlaprevir SCH900518 (Schering/Merck), ABT-450(Abbott/Enanta), ACH-1625 (Achillion), BI 201335 (Boehringer Ingelheim),PHX1766 (Phenomix), VX-500 (Vertex) and telaprevir (VX-950) (Vertex).Further examples of protease inhibitors include substrate-based NS3protease inhibitors (Attwood et al., Antiviral peptide derivatives, PCTWO 98/22496, 1998; Attwood et al, Antiviral Chemistry and Chemotherapy1999, 10, 259-273; Attwood et al., Preparation and use of amino acidderivatives as anti-viral agents, German Patent Pub. DE 19914474; Tunget al., Inhibitors of serine proteases, particularly hepatitis C virusNS3 protease, PCT WO 98/17679), including alphaketoamides andhydrazinoureas, and inhibitors that terminate in an electrophile such asa boronic acid or phosphonate (Llinas-Brunet et al, Hepatitis Cinhibitor peptide analogues, PCT WO 99/07734); Non-substrate-based NS3protease inhibitors such as 2,4,6-trihydroxy-3-nitro-benzamidederivatives (Sudo K. et al, Biochemical and Biophysical ResearchCommunications, 1997, 238, 643-647; Sudo K. et al., Antiviral Chemistryand Chemotherapy, 1998, 9, 186), including RD3-4082 and RD3-4078, theformer substituted on the amide with a 14 carbon chain and the latterprocessing a para-phenoxyphenyl group; and Sch 68631, aphenanthrenequinone, an HCV protease inhibitor (Chu M. et al.,Tetrahedron Letters 37:7229-7232, 1996).

SCH 351633, isolated from the fungus Penicillium griscofulvum, wasidentified as a protease inhibitor (Chu M., et al., Bioorganic andMedicinal Chemistry Letters 9: 1949-1952). Eglin c, isolated from leech,is a potent inhibitor of several serine proteases such as S. griseusproteases A and B, a-chymotrypsin, chymase and subtilisin. Qasim M. A.et al., Biochemistry 36: 1598-1607, 1997.

U.S. patents disclosing protease inhibitors for the treatment of HCVinclude, for example, U.S. Pat. No. 6,004,933 to Spruce et al., whichdiscloses a class of cysteine protease inhibitors for inhibiting HCVendopeptidase 2; U.S. Pat. No. 5,990,276 to Zhang et al., whichdiscloses synthetic inhibitors of hepatitis C virus NS3 protease; U.S.Pat. No. 5,538,865 to Reyes et a; WO 02/008251 to Corvas International,Inc, and U.S. Pat. No. 7,169,760, US2005/176648, WO 02/08187 and WO02/008256 to Schering Corporation. HCV inhibitor tripeptides aredisclosed in U.S. Pat. Nos. 6,534,523, 6,410,531, and 6,420,380 toBoehringer Ingelheim and WO 02/060926 to Bristol Myers Squibb. Diarylpeptides as NS3 serine protease inhibitors of HCV are disclosed in WO02/48172 and U.S. Pat. No. 6,911,428 to Schering Corporation.Imidazoleidinones as NS3 serine protease inhibitors of HCV are disclosedin WO 02/08198 and U.S. Pat. No. 6,838,475 to Schering Corporation andWO 02/48157 and U.S. Pat. No. 6,727,366 to Bristol Myers Squibb. WO98/17679 and U.S. Pat. No. 6,265,380 to Vertex Pharmaceuticals and WO02/48116 and U.S. Pat. No. 6,653,295 to Bristol Myers Squibb alsodisclose HCV protease inhibitors. Further examples of HCV serineprotease inhibitors are provided in U.S. Pat. No. 6,872,805(Bristol-Myers Squibb); WO 2006000085 (Boehringer Ingelheim); U.S. Pat.No. 7,208,600 (Vertex); US 2006/0046956 (Schering-Plough); WO2007/001406 (Chiron); US 2005/0153877; WO 2006/119061 (Merck); WO00/09543 (Boehringer Ingelheim), U.S. Pat. No. 6,323,180 (BoehringerIngelheim) WO 03/064456 (Boehringer Ingelheim), U.S. Pat. No. 6,642,204(Boehringer Ingelheim), WO 03/064416 (Boehringer ingelheim), U.S. Pat.No. 7,091,184 (Boehringer Ingelheim), WO 03/053349 (Bristol-MyersSquibb), U.S. Pat. No. 6,867,185, WO 03/099316 (Bristol-Myers Squibb),U.S. Pat. No. 6,869,964, WO 03/099274 (Bristol-Myers Squibb), U.S. Pat.No. 6,995,174, WO 2004/032827 (Bristol-Myers Squibb), U.S. Pat. No.7,041,698, WO 2004/043339 and U.S. Pat. No. 6,878,722 (Bristol-MyersSquibb).

Thiazolidine derivatives which show relevant inhibition in areverse-phase HPLC assay with an NS3/4A fusion protein and NS5A/5Bsubstrate (Sudo K. et al, Antiviral Research, 1996, 32, 9-18),especially compound RD-1-6250, possessing a fused cinnamoyl moietysubstituted with a long alkyl chain, RD4 6205 and RD4 6193;

Thiazolidines and benzanilides identified in Kakiuchi N. et al, J. EBSLetters 421, 217-220; Takeshita N. et al, Analytical Biochemistry, 1997,247, 242-246;

A phenanthrenequinone possessing activity against protease in a SDS-PAGEand autoradiography assay isolated from the fermentation culture brothof Streptomyces sp., SCH 68631 (Chu M. et al, Tetrahedron Letters, 1996,37, 7229-7232), and SCH 351633, isolated from the fungus Penicilliumgriseofulvum, which demonstrates activity in a scintillation proximityassay (Chu M. et al, Bioorganic and Medicinal Chemistry Letters 9,1949-1952);

Helicase inhibitors (Diana G. D. et al, Compounds, compositions andmethods for treatment of hepatitis C, U.S. Pat. No. 5,633,358; Diana G.D. et al, Piperidine derivatives, pharmaceutical compositions thereofand their use in the treatment of hepatitis C, PCT WO 97/36554);

HCV polymerase inhibitors, including nucleoside and non-nucleosidepolymerase inhibors, such as ribavirin, viramidine, clemizole, filibuvir(PF-00868554), HCV POL, NM-283 (valopicitabine), MK-0608,7-Fluoro-MK-0608, MK-3281, IDX-375. ABT-072, ABT-333, ANA598, BI 207127,GS 9190, PSI-6130, R1626, PSI-6206, PSI-35938, PSI-7851, PSI-7977,RG1479, RG7128, HCV-796 VCH-759 or VCH-916, and salts and prodrugsthereof.

Gliotoxin (Ferrari R. et al, Journal of Virology, 1999, 73, 1649-1654),and the natural product cerulenin (Lohmann V. et al., Virology, 1998,249, 108-118);

Interfering RNA (iRNA) based antivirals, including short interfering RNA(siRNA) based antivirals, such as Sirna-034 and others described inInternational Patent Publication Nos. WO/03/070750 and WO 2005/012525,and U.S. Patent Publication No. US 2004/0209831.

Antisense phosphorothioate oligodeoxynucleotides (S-ODN) complementaryto sequence stretches in the 5′ non-coding region (NCR) of the virus(Alt M. et al., Hepatology, 1995, 22, 1 ‘O’-717), or nucleotides 326-348comprising the 3′ end of the NCR and nucleotides 371-388 located in thecore coding region of the HCV RNA (Alt M. et al, Archives of Virology,1997, 142, 589-599; Galderisi U. et al, Journal of Cellular Physiology,1999, 181, 251-257);

Inhibitors of IRES-dependent translation (Ikeda N et al., Agent for theprevention and treatment of hepatitis C, Japanese Patent Pub.JP-08268890; Kai Y. et al, Prevention and treatment of viral diseases,Japanese Patent Pub. JP-10101591);

HCV entry inhibitors, such as celgosivir (MK-3253) (MIGENIX Inc.), SP-30(Samaritan Pharmaceuticals), ITX4520 (iTherX), ITX5061 (iTherX), PRO-206(Progenies Pharmaceuticals) and other entry inhibitors by ProgeniesPharmaceuticals, e.g., as disclosed in U.S. Patent Publication No.2006/0198855.

Ribozymes, such as nuclease-resistant ribozymes (Maccjak, D. J. et al,Hepatology 1999, 30, abstract 995) and those disclosed in U.S. Pat. No.6,043,077 to Barber et al, and U.S. Pat. Nos. 5,869,253 and 5,610,054 toDraper et al; and

Nucleoside analogs have also been developed for the treatment ofFlaviviridae infections.

In certain embodiments, the compounds provided herein can beadministered in combination with any of the compounds described byIdenix Pharmaceuticals in International Publication Nos. WO 01/90121, WO01/92282, WO 2004/003000, 2004/002422, WO 2004/002999, WO 10/014134 andWO 11/123586.

Other patent applications disclosing the use of certain nucleosideanalogs that can be used as second agents to treat hepatitis C virusinclude: PCT/CAOO/01316 (WO 01/32153; filed Nov. 3, 2000) andPCT/CA01/00197 (WO 01/60315; filed Feb. 19, 2001) filed by BioChemPharma, inc. (now Shire Biochem, Inc.); PCT/US02/01531 (WO 02/057425;filed Jan. 18, 2002); PCT/US02/03086 (WO 02/057287; filed Jan. 18,2002); U.S. Pat. Nos. 7,202,224; 7,125,855; 7,105,499 and 6,777,395 byMerck & Co., Inc.; PCT/EP01/09633 (WO 02/18404; published Aug. 21,2001); US 2006/0040890; 2005/0038240; 2004/0121980; 6,846,810; 6,784,166and 6,660,721 by Roche; PCT Publication Nos. WO 01/79246 (filed Apr. 13,2001), WO 02/32920 (filed Oct. 18, 2001), WO 02/48165, WO 05/003147; US2005/0009737; US 2005/0009737; 7,094,770, 6,927,291, WO 08/12163434, WO10/077554, WO 09/152095, WO 10/075549, and WO 10/135569 by Pharmasset,Ltd.

Further compounds that can be used as second agents to treat hepatitis Cvirus are disclosed in PCT Publication No. WO 99/43691 to EmoryUniversity, entitled “2′-Fluoronucleosides”. The use of certain2′-fluoronucleosides to treat HCV is disclosed.

Other miscellaneous compounds that can be used as second agents include1-amino-alkylcyclohexanes (U.S. Pat. No. 6,034,134 to Gold et al.),alkyl lipids (U.S. Pat. No. 5,922,757 to Chojkier et al.), vitamin E andother antioxidants (U.S. Pat. No. 5,922,757 to Chojkier et al.),squalene, amantadine, bile acids (U.S. Pat. No. 5,846,964 to Ozeki etal.), N-(phosphonoacetyl)-L-aspartic acid, (U.S. Pat. No. 5,830,905 toDiana et al.), benzenedicarboxamides (U.S. Pat. No. 5,633,388 to Dianaet al.), polyadenylic acid derivatives (U.S. Pat. No. 5,496,546 to Wanget al.), 2′,3′-dideoxyinosine (U.S. Pat. No. 5,026,687 to Yarchoan etal.), benzimidazoles (U.S. Pat. No. 5,891,874 to Colacino et al.), plantextracts (U.S. Pat. No. 5,837,257 to Tsai et al., U.S. Pat. No.5,725,859 to Omer et al., and U.S. Pat. No. 6,056,961), and piperidenes(U.S. Pat. No. 5,830,905 to Diana et al.).

Exemplary Additional Therapeutic Agents for Treatment of HCV

In one embodiment, one or more compounds provided herein can beadministered in combination or alternation with an anti-hepatitis Cvirus interferon, such as Intron A® (interferon alfa-2b) and Pegasys®(Peginterferon alfa-2a); Roferon A® (Recombinant interferon alfa-2a),Infergen® (consensus interferon; interferon alfacon-1), PEG-Intron®(pegylated interferon alfa-2b) and Pegasys® (pegylated interferonalfa-2a), optionally in further combination with ribavirin.

In one embodiment, the anti-hepatitis C virus interferon is infergen,IL-29 (PEG-Interferon lambda), R7025 (Maxy-alpha), Belerofon, OralTnterferon alpha, BLX-883 (Locteron), omega interferon, multiferon,medusa interferon, Albuferon or REBIF®.

In one embodiment, one or more compounds provided herein can beadministered in combination or alternation with an anti-hepatitis Cvirus polymerase inhibitor, such as ribavirin, viramidine, HCV POL,NM-283 (valopicitabine), PSI-7977, PSI-938, MK-0608, 7-Fluoro-MK-0608,PSI-6130, R1626, IDX-184, INX-189, PSI-6206, PSI-35938, R1479, HCV-796or R7128.

In one embodiment, one or more compounds provided herein can beadministered in combination or alternation with an anti-HCVproteaseinhibitor such as ITMN-191, SCH 503034, VX950 (telaprevir), GNS-227, orMedivir HCV Protease Inhibitor.

In one embodiment, one or more compounds provided herein can beadministered in combination or alternation with an anti-HCV vaccine,such as TG4040, PeviPROTM, CGI-5005, HCV/MF59, GV1001, IC41 or INNO0101(E1).

In one embodiment, one or more compounds provided herein can beadministered in combination or alternation with an anti-HCV monoclonalantibody, such as AB68 or XTL-6865 (formerly HepX-C); or ananti-hepatitis C virus polyclonal antibody, such as cicavir.

In one embodiment, one or more compounds provided herein can beadministered in combination or alternation with an anti-hepatitis Cvirus immunomodulator, such as Zadaxin® (thymalfasin), NOV-205 orOglufanide.

In one embodiment, one or more compounds provided herein can beadministered in combination or alternation with Nexavar, doxorubicin,PI-88, amantadine, JBK-122, VGX-4 IOC, MX-3253 (Ceglosivir), Suvus(BIVN-401 or virostat), PF-03491390 (formerly IDN-6556), G126270,UT-231B, DEBIO-025, EMZ702, ACH-0137171, MitoQ, ANA975, AVI-4065,Bavituxinab (Tarvacin), Alinia (nitrazoxanide) or PYN17.

Prodrug Forms

The 5′-hydroxyl moiety in the nucleosides described herein, and hydroxygroups on the JAK inhibitors described herein, can be modified to be inprodrug form. For example, the 5′-hydroxy in nucleosides can be replacedwith a 5′-OR¹ moiety, where R¹ is an optionally substituted alkyl, anoptionally substituted cycloalkyl, an optionally substituted aralkyl,dialkylaminoalkylene, alkyl-C(═O)—, aryl-C(═O)—, alkoxyalkyl-C(═O)—,aryloxyalkyl-C(═O)—, alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, an—O-linked amino acid, diphosphate, triphosphate or derivatives thereof,or

wherein:

V¹ is O or S;

R¹⁰ is selected from O⁻, —OH, an optionally substituted aryloxy orheteroaryl-oxy-, alkyl-C(═O)—O—CH₂—O—, alkyl-C(═O)—S—CH₂CH₂—O—,pivaloyloxymethyl, —NH—CH₂-aryl, —O—CH₂—O—C(O)—OR^(a1), an —N-linkedamino acid, an —N-linked amino acid ester,

or OR¹ can be

where Ar¹ is selected from phenyl, pyridinyl, monocyclic heteroaryl,substituted phenyl with 1-3 substituents, and monoheterocyclicheteroaryl with 1-2 substitutents, wherein each substituent isindependently selected from the group consisting of —F, —Cl, —Br, —I,C₁₋₆ alkyl, —CF₃, —OMe, —NMe₂, —OEt, —CO₂R^(a1), —CONH₂, —SMe,—S(═O)₂Me, —S(═O)₂NH₂, and CN;

or R¹ and R¹⁰ can combine to form a cyclic phosphate of the formula:

where R¹⁹ is selected from N-linked amino acid ester, OR^(a1) or OR²⁰,wherein R²⁰ is substituted aryl with 1-3 substituents, or substitutedheteroaryl with 1-2 substituents, wherein each substituent isindependently selected from R^(a1) and R^(d1).

R¹¹ is selected from O⁻, —OH, an optionally substituted aryloxy oraryl-O—, alkyl-C(═O)—O—CH₂—O—, alkyl-C(═O)—S—CH₂CH₂—O—,pivaloyloxymethyl, —NH—CH₂-aryl, —O—CH₂—O—C(O)—OR^(a1), an —N-linkedamino acid, an —N-linked amino acid ester,

or OR¹ can be

where Ar¹ is selected from phenyl, pyridinyl, monocyclic heteroaryl,substituted phenyl with 1-3 substituents, and monoheterocyclicheteroaryl with 1-2 substitutents, wherein each substituent isindependently selected from the group consisting of —F, —Cl, —Br, —I,C₁₋₆ alkyl, —CF₃, —OMe, —NMe₂, —OEt, —CO₂R^(a1), —CONH₂, —SMe,—S(═O)₂Me, —S(═O)₂NH₂, and CN;

or R¹ and R¹⁰ can combine to form a cyclic phosphate of the formula:

where R¹⁹ is selected from N-linked amino acid ester, OR^(a1) or OR²⁰,wherein R²⁰ is substituted aryl with 1-3 substituents, or substitutedheteroaryl with 1-2 substituents, wherein each substituent isindependently selected from R^(a1) and R^(d1),

each R¹² and R¹³ are, independently, —C—N or an optionally substitutedsubstituent selected from C₁₋₈ organylcarbonyl, C₁₋₈ alkoxycarbonyl andC₁₋₈ organylaminocarbonyl;

each R¹⁴ is hydrogen or an optionally substituted C₁₋₆ alkyl;

each m is independently 1 or 2, and if both R¹⁰ and R¹¹ are

each R¹², each R¹³, each R¹⁴ and each m can be the same or different.

R^(a1), R^(b1), R^(c1), and R^(d1) are each independently selected fromhydrogen, an optionally substituted alkyl, an optionally substitutedalkenyl, an optionally substituted alkynyl, an optionally substitutedaryl, an optionally substituted heteroaryl, an optionally substitutedaralkyl and an optionally substituted heteroaryl-(C₁₋₆ alkyl).

In one embodiment, R¹ is a mono-phosphate, di-phosphate, tri-phosphate,or phosphate prodrug.

V. Pharmaceutical Compositions

Humans suffering from effects caused by any of the diseases describedherein, and in particular, HIV infection, can be treated byadministering to the patient an effective amount of the compositionsdescribed above, in the presence of a pharmaceutically acceptablecarrier or diluent, for any of the indications or modes ofadministration as described in detail herein. The active materials canbe administered by any appropriate route, for example, orally,parenterally, enterally, intravenously, intradermally, subcutaneously,transdermally, intranasally or topically, in liquid or solid form.

The active compounds are included in the pharmaceutically acceptablecarrier or diluent in an amount sufficient to deliver to a patient atherapeutically effective amount of compound to inhibit viralpropagation in vivo, especially HIV propagation, without causing serioustoxic effects in the treated patient. While not wishing to be bound to aparticular theory, it is believed that the JAK inhibitors render thecellular milieu non-supportive of productive replication. By “inhibitoryamount” is meant an amount of active ingredient sufficient to exert aninhibitory effect as measured by, for example, an assay such as the onesdescribed herein.

A preferred dose of the compound for all the above-mentioned conditionswill be in the range from about 1 to 75 mg/kg, preferably 1 to 20 mg/kg,of body weight per day, more generally 0.1 to about 100 mg per kilogrambody weight of the recipient per day. The effective dosage range of thepharmaceutically acceptable derivatives can be calculated based on theweight of the parent nucleoside or other agent to be delivered. If thederivative exhibits activity in itself, the effective dosage can beestimated as above using the weight of the derivative, or by other meansknown to those skilled in the art.

The compounds are conveniently administered in unit any suitable dosageform, including but not limited to one containing 7 to 3,000 mg,preferably 70 to 1,400 mg of active ingredient per unit dosage form. Anoral dosage of 50 to 1,000 mg is usually convenient.

Ideally, the active ingredient should be administered to achieve peakplasma concentrations of the active compound of from about 0.02 to 70micromolar, preferably about 0.5 to 10 micromolar. This may be achieved,for example, by the intravenous injection of a 0.1 to 25% solution ofthe active ingredient, optionally in saline, or administered as a bolusof the active ingredient.

The concentration of active compound in the drug composition will dependon absorption, distribution, metabolism and excretion rates of the drugas well as other factors known to those of skill in the art. It is to benoted that dosage values will also vary with the severity of thecondition to be alleviated. It is to be further understood that for anyparticular subject, specific dosage regimens should be adjusted overtime according to the individual need and the professional judgment ofthe person administering or supervising the administration of thecompositions, and that the concentration ranges set forth herein areexemplary only and are not intended to limit the scope or practice ofthe claimed composition. The active ingredient may be administered atonce, or may be divided into a number of smaller doses to beadministered at varying intervals of time.

A preferred mode of administration of the active compound is oral. Oralcompositions will generally include an inert diluent or an ediblecarrier. They may be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules. Pharmaceutically compatible bind agents,and/or adjuvant materials can be included as part of the composition.

The tablets, pills, capsules, troches and the like can contain any ofthe following ingredients, or compounds of a similar nature: a bindersuch as microcrystalline cellulose, gum tragacanth or gelatin; anexcipient such as starch or lactose, a disintegrating agent such asalginic acid, Primogel, or corn starch; a lubricant such as magnesiumstearate or Sterotes; a glidant such as colloidal silicon dioxide; asweetening agent such as sucrose or saccharin; or a flavoring agent suchas peppermint, methyl salicylate, or orange flavoring. When the dosageunit form is a capsule, it can contain, in addition to material of theabove type, a liquid carrier such as a fatty oil. In addition, dosageunit forms can contain various other materials which modify the physicalform of the dosage unit, for example, coatings of sugar, shellac, orother enteric agents.

The compounds can be administered as a component of an elixir,suspension, syrup, wafer, chewing gum or the like. A syrup may contain,in addition to the active compounds, sucrose as a sweetening agent andcertain preservatives, dyes and colorings and flavors.

The compounds or their pharmaceutically acceptable derivative or saltsthereof can also be mixed with other active materials that do not impairthe desired action, or with materials that supplement the desiredaction, such as antibiotics, antifungals, antiinflammatories, proteaseinhibitors, or other nucleoside or non-nucleoside antiviral agents, asdiscussed in more detail above. Solutions or suspensions used forparental, intradermal, subcutaneous, or topical application can includethe following components: a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates and agents for theadjustment of tonicity such as sodium chloride or dextrose. The parentalpreparation can be enclosed in ampoules, disposable syringes or multipledose vials made of glass or plastic.

If administered intravenously, preferred carriers are physiologicalsaline or phosphate buffered saline (PBS).

Liposomal suspensions (including liposomes targeted to infected cellswith monoclonal antibodies to viral antigens) are also preferred aspharmaceutically acceptable carriers, these may be prepared according tomethods known to those skilled in the art, for example, as described inU.S. Pat. No. 4,522,811. For example, liposome formulations may beprepared by dissolving appropriate lipid(s) (such as stearoylphosphatidyl ethanolamine, stearoyl phosphatidyl choline, arachadoylphosphatidyl choline, and cholesterol) in an inorganic solvent that isthen evaporated, leaving behind a thin film of dried lipid on thesurface of the container. An aqueous solution of the active compound orits monophosphate, diphosphate, and/or triphosphate derivatives is thenintroduced into the container. The container is then swirled by hand tofree lipid material from the sides of the container and to disperselipid aggregates, thereby forming the liposomal suspension.

In one embodiment, the composition is a co-formulated pill, tablet, orother oral drug delivery vehicle including one or more of the JAKinhibitors described herein, and optionally including one or moreadditional antiviral agents.

In another embodiment, the JAK inhibitors described herein areco-formulated with ATRIPLA® (efavirenz 600 mg/emtricitabine [(−)-FTC]200 mg/tenofovir disoproxil fumarate 300 mg), and, optionally, with athymidine nRTI such as AZT and a guanine nRTI (or a compound such asDAPD which is deaminated in vivo to form a guanine nRTI, in this case,DXG). Because efavirenz is an NNRTI, tenofovir is an adenine nRTI,(−)-FTC is a cytosine nRTI, and AZT is a thymidine nRTI, and DAPD isdeaminated in vivo to form DXG (a guanine NRTI), the combination of thecoformulated compounds will provide, in addition to the JAK inhibitors,all four bases (ACTG) plus an additional agent capable of interactingwith HIV in a different mechanism.

Controlled Release Formulations

All of the U.S. patents cited in this section on controlled releaseformulations are incorporated by reference in their entirety.

The field of biodegradable polymers has developed rapidly since thesynthesis and biodegradability of polylactic acid was reported byKulkarni et al., in 1966 (“Polylactic acid for surgical implants,” Arch.Surg., 93:839). Examples of other polymers which have been reported asuseful as a matrix material for delivery devices include polyanhydrides,polyesters such as polyglycolides and polylactide-co-glycolides,polyamino acids such as polylysine, polymers and copolymers ofpolyethylene oxide, acrylic terminated polyethylene oxide, polyamides,polyurethanes, polyorthoesters, polyacrylonitriles, andpolyphosphazenes. See, for example, U.S. Pat. Nos. 4,891,225 and4,906,474 to Langer (polyanhydrides), U.S. Pat. No. 4,767,628 toHutchinson (polylactide, polylactide-co-glycolide acid), and U.S. Pat.No. 4,530,840 to Tice, et al. (polylactide, polyglycolide, andcopolymers). See also U.S. Pat. No. 5,626,863 to Hubbell, et al whichdescribes photopolymerizable biodegradable hydrogels as tissuecontacting materials and controlled release carriers (hydrogels ofpolymerized and crosslinked macromers comprising hydrophilic oligomershaving biodegradable monomeric or oligomeric extensions, which are endcapped monomers or oligomers capable of polymerization andcrosslinking); and PCT WO 97/05185 filed by Focal. Inc. directed tomultiblock biodegradable hydrogels for use as controlled release agentsfor drug delivery and tissue treatment agents.

Degradable materials of biological origin are well known, for example,crosslinked gelatin. Hyaluronic acid has been crosslinked and used as adegradable swelling polymer for biomedical applications (U.S. Pat. No.4,957,744 to Della Valle et. al.; (1991) “Surface modification ofpolymeric biomaterials for reduced thrombogenicity,” Polym. Mater. Sci.Eng., 62:731735]).

Many dispersion systems are currently in use as, or being explored foruse as, carriers of substances, particularly biologically activecompounds. Dispersion systems used for pharmaceutical and cosmeticformulations can be categorized as either suspensions or emulsions.Suspensions are defined as solid particles ranging in size from a fewmanometers up to hundreds of microns, dispersed in a liquid medium usingsuspending agents. Solid particles include microspheres, microcapsules,and nanospheres. Emulsions are defined as dispersions of one liquid inanother, stabilized by an interfacial film of emulsifiers such assurfactants and lipids. Emulsion formulations include water in oil andoil in water emulsions, multiple emulsions, microemulsions,microdroplets, and liposomes. Microdroplets are unilamellar phospholipidvesicles that consist of a spherical lipid layer with an oil phaseinside, as defined in U.S. Pat. Nos. 4,622,219 and 4,725,442 issued toHaynes. Liposomes are phospholipid vesicles prepared by mixingwater-insoluble polar lipids with an aqueous solution. The unfavorableentropy caused by mixing the insoluble lipid in the water produces ahighly ordered assembly of concentric closed membranes of phospholipidwith entrapped aqueous solution.

U.S. Pat. No. 4,938,763 to Dunn, et al., discloses a method for formingan implant in situ by dissolving a nonreactive, water insolublethermoplastic polymer in a biocompatible, water soluble solvent to forma liquid, placing the liquid within the body, and allowing the solventto dissipate to produce a solid implant. The polymer solution can beplaced in the body via syringe. The implant can assume the shape of itssurrounding cavity. In an alternative embodiment, the implant is formedfrom reactive, liquid oligomeric polymers which contain no solvent andwhich cure in place to form solids, usually with the addition of acuring catalyst.

A number of patents disclose drug delivery systems that can be used toadminister the combination of the thymidine and non-thymidine nucleosideantiviral agents, or prodrugs thereof. U.S. Pat. No. 5,749,847 disclosesa method for the delivery of nucleotides into organisms byelectrophoration. U.S. Pat. No. 5,718,921 discloses microspherescomprising polymer and drug dispersed there within. U.S. Pat. No.5,629,009 discloses a delivery system for the controlled release ofbioactive factors. U.S. Pat. No. 5,578,325 discloses nanoparticles andmicroparticles of non-linear hydrophilic hydrophobic multiblockcopolymers. U.S. Pat. No. 5,545,409 discloses a delivery system for thecontrolled release of bioactive factors. U.S. Pat. No. 5,494,682discloses ionically cross-linked polymeric microcapsules.

U.S. Pat. No. 5,728,402 to Andrx Pharmaceuticals, Inc. describes acontrolled release formulation that includes an internal phase whichcomprises the active drug, its salt or prodrug, in admixture with ahydrogel forming agent, and an external phase which comprises a coatingwhich resists dissolution in the stomach. U.S. Pat. Nos. 5,736,159 and5,558,879 to Andrx Pharmaceuticals, Inc. discloses a controlled releaseformulation for drugs with little water solubility in which a passagewayis formed in situ. U.S. Pat. No. 5,567,441 to Andrx Pharmaceuticals,Inc. discloses a once-a-day controlled release formulation. U.S. Pat.No. 5,508,040 discloses a multiparticulate pulsatile drug deliverysystem. U.S. Pat. No. 5,472,708 discloses a pulsatile particle baseddrug delivery system. U.S. Pat. No. 5,458,888 describes a controlledrelease tablet formulation which can be made using a blend having aninternal drug containing phase and an external phase which comprises apolyethylene glycol polymer which has a weight average molecular weightof from 3,000 to 10,000. U.S. Pat. No. 5,419,917 discloses methods forthe modification of the rate of release of a drug form a hydrogel whichis based on the use of an effective amount of a pharmaceuticallyacceptable ionizable compound that is capable of providing asubstantially zero-order release rate of drug from the hydrogel. U.S.Pat. No. 5,458,888 discloses a controlled release tablet formulation.

U.S. Pat. No. 5,641,745 to Elan Corporation, plc discloses a controlledrelease pharmaceutical formulation which comprises the active drug in abiodegradable polymer to form microspheres or nanospheres. Thebiodegradable polymer is suitably poly-D,L-lactide or a blend ofpoly-D,L-lactide and poly-D,L-lactide-co-glycolide. U.S. Pat. No.5,616,345 to Elan Corporation plc describes a controlled absorptionformulation for once a day administration that includes the activecompound in association with an organic acid, and a multi-layer membranesurrounding the core and containing a major proportion of apharmaceutically acceptable film-forming, water insoluble syntheticpolymer and a minor proportion of a pharmaceutically acceptablefilm-forming water soluble synthetic polymer. U.S. Pat. No. 5,641,515discloses a controlled release formulation based on biodegradablenanoparticles. U.S. Pat. No. 5,637,320 discloses a controlled absorptionformulation for once a day administration. U.S. Pat. Nos. 5,580,580 and5,540,938 are directed to formulations and their use in the treatment ofneurological diseases. U.S. Pat. No. 5,533,995 is directed to a passivetransdermal device with controlled drug delivery. U.S. Pat. No.5,505,962 describes a controlled release pharmaceutical formulation.

Prodrug Formulations

The JAK inhibitors, as well as the nucleosides or other compounds whichare described herein for use in combination or alternation therapy withthe JAK inhibitors or their related compounds, can be administered as anacylated prodrug or a nucleotide prodrug, as described in detail below.

Any of the JAK inhibitors, nucleosides, or other compounds describedherein that contain a hydroxyl or amine function can be administered asa nucleotide prodrug to increase the activity, bioavailability,stability or otherwise alter the properties of the nucleoside. A numberof nucleotide prodrug ligands are known. In general, alkylation,acylation or other lipophilic modification of the hydroxyl group of thecompound or of the mono, di or triphosphate of the nucleoside willincrease the stability of the nucleotide. Examples of substituent groupsthat can replace one or more hydrogens on the phosphate moiety orhydroxyl are alkyl, aryl, steroids, carbohydrates, including sugars,1,2-diacylglycerol and alcohols. Many are described in R. Jones and N.Bischoberger, Antiviral Research, 27 (1995) 1 17. Any of these can beused in combination with the disclosed nucleosides or other compounds toachieve a desire effect.

The active nucleoside or other hydroxyl containing compound can also beprovided as an ether lipid (and particularly a 5′-ether lipid for anucleoside), as disclosed in the following references, Kucera, L. S., N.Iyer, E. Leake, A. Raben, Modest E. K., D. L. W., and C. Piantadosi.1990. “Novel membrane-interactive ether lipid analogs that inhibitinfectious HIV-1 production and induce defective virus formation.” AIDSRes. Hum. Retroviruses. 6:491 501; Piantadosi, C., J. Marasco C. J., S.L. Morris-Natschke, K. L. Meyer, F. Gumus, J. R. Surles, K. S. Ishaq, L.S. Kucera, N. Iyer, C. A. Wallen, S. Piantadosi, and E. J. Modest. 1991.“Synthesis and evaluation of novel ether lipid nucleoside conjugates foranti-HIV activity.” J. Med. Chem. 34:1408.1414; Hosteller, K. Y., D. D.Richrnan, D. A. Carson, L. M. Stuhmillcr, G. M. T. van Wijk, and H. vanden Bosch. 1992. “Greatly enhanced inhibition of human immunodeficiencyvirus type 1 replication in CEM and HT4-6C cells by 3′-deoxythymidinediphosphate dimyristoylglycerol, a lipid prodrug of 3′-deoxythymidine.”Antimicrob. Agents Chemother. 36:2025.2029; Hostetler, K. Y., L. M.Stuhmiller, H. B. Lenting, H. van den Bosch, and D. D. Richman, 1990.“Synthesis and antiretroviral activity of phospholipid analogs ofazidothymidine and other antiviral nucleosides.” J. Biol. Chem.265:61127.

Nonlimiting examples of U.S. patents that disclose suitable lipophilicsubstituents that can be covalently incorporated into the nucleoside orother hydroxyl or amine containing compound, preferably at the 5′-OHposition of the nucleoside or lipophilic preparations, include U.S. Pat.No. 5,149,794 (Sep. 22, 1992, Yatvin et al.); U.S. Pat. No. 5,194,654(Mar. 16, 1993, Hostetler et al., U.S. Pat. No. 5,223,263 (Jun. 29,1993, Hostetler et al.); U.S. Pat. No. 5,256,641 (Oct. 26, 1993, Yatvinet al.); U.S. Pat. No. 5,411,947 (May 2, 1995, Hostetler et al.); U.S.Pat. No. 5,463,092 (Oct. 31, 1995, Hostetler et al.); U.S. Pat. No.5,543,389 (Aug. 6, 1996, Yatvin et al.); U.S. Pat. No. 5,543,390 (Aug.6, 1996, Yatvin et al.); U.S. Pat. No. 5,543,391 (Aug. 6, 1996, Yatvinet al.); and U.S. Pat. No. 5,554,728 (Sep. 10, 1996; Basava et-al.),Foreign patent applications that disclose lipophilic substituents thatcan be attached to the nucleosides of the present invention, orlipophilic preparations, include WO 89/02733, WO 90/00555, WO 91/16920,WO 91/18914, WO 93/00910, WO 94/26273, WO 96/15132, EP 0 350 287, EP93917054.4, and WO 91/19721.

Nonlimiting examples of nucleotide prodrugs are described in thefollowing references: Ho, D. H. W. (1973) “Distribution of Kinase anddeaminase of 1β-D-arabinofuranosylcytosine in tissues of man and muse.”Cancer Res. 33, 2816 2820; Holy, A. (1993) Isopolar phosphorous-modifiednucleotide analogues,” In: De Clereq (Ed.), Advances in Antiviral DrugDesign, Vol. I, JAI Press, pp. 179 231; Hong, C. I., Nechaev, A., andWest, C. R. (1979a) “Synthesis and antitumor activity of1l-D-arabino-furanosylcytosine conjugates of cortisol and cortisone.”Biochem. Biophys. Rs. Commun. 88, 1223 1229; Hong, C. I., Nechaev, A.,Kirisits, A. J. Buchheit, D. J. and West, C. R. (1980) “Nucleosideconjugates as potential antitumor agents. 3. Synthesis and antitumoractivity of 1-(β-D-arabinofuranosyl)cytosine conjugates ofcorticosteroids and selected lipophilic alcohols.” J. Med. Chem. 28, 171177; Hosteller, K. Y., Stuhmiller, L. M., Lenting, H. B. M. van denBosch, H. and Richman J Biol. Chem. 265, 6112 6117; Hosteller, K. Y.,Carson, D. A. and Richman, D. D. (1991); “Phosphatidylazidothymidine:mechanism of antiretroviral action in CEM cells.” J. Biol Chem. 266,11714 11717; Hosteller, K. Y., Korba, B. Sridhar, C., Gardener, M.(1994a) “Antiviral activity of phosphatidyl-dideoxycytidine in hepatitisB-infected cells and enhanced hepatic uptake in mice.” Antiviral Res.24, 59 67; Hosteller, K. Y., Richman, D. D., Sridhar. C. N. Felgner, P.L. Felgner, J., Ricci, J., Gardener, M. F. Selleseth, D. W. and Ellis,M. N. (1994b) “Phosphatidylazidothymidine and phosphatidyl-ddC:Assessment of uptake in mouse lymphoid tissues and antiviral activitiesin human immunodeficiency virus-infected cells and in rauscher leukemiavirus-infected mice.” Antimicrobial Agents Chemother. 38, 2792 2797;Hunston, R. N., Jones, A. A. McGuigan, C., Walker, R. T., Balzarini, J.,and DeClercq, E. (1984) “Synthesis and biological properties of somecyclic phosphotriesters derived from 2′-deoxy-5-fluorouridine.” J. Med.Chem. 27, 440 444; Ji, Y. H., Moog, C., Schmitt, G., Bischoff, P. andLuu, B. (1990); “Monophosphoric acid esters of 7-β-hydroxycholesteroland of pyrimidine nucleoside as potential antitumor agents: synthesisand preliminary evaluation of antitumor activity.” J. Med. Chem. 33 22642270; Jones, A. S., McGuigan, C., Walker, R. T., Balzarini, J. andDeClercq, E. (1984) “Synthesis, properties, and biological activity ofsome nucleoside cyclic phosphoramidates.” J. Chem. Soc. Perkin Trans. I,1471 1474; Juodka, B. A. and Smart, J. (1974) “Synthesis ofdiribonucleoside phosph (P.fwdarw.N) amino acid derivatives.” Coll.Czech. Chem. Comm. 39, 363 968; Kataoka, S., Imai, J., Yamaji, N., Kato,M., Saito, M., Kawada, T. and Imai, S. (1989) “Alkylated cAMPderivatives; selective synthesis and biological activities.” NucleicAcids Res. Sym. Ser. 21, 1 2; Kataoka, S., Uchida, “(cAMP) benzyl andmethyl triesters.” Heterocycles 32, 1351 1356; Kinchington, D., Harvey,J. J., O'Connor, T. J., Jones, B. C. N. M., Devine, K. G.,Taylor-Robinson D., Jeffries, D. J. and McGuigan, C. (1992) “Comparisonof antiviral effects of zidovudine phosphoramidate andphosphorodiamidate derivatives against HIV and ULV in vitro.” AntiviralChem. Chemother. 3, 107 112; Kodama, K., Morozumi, M., Saithoh, K. T.,Kuninaka, H., Yosino, H. and Saneyoshi, M. (1989) “Antitumor activityand pharmacology of 1-β-D-arabinofuranosylcytosine-5′-stearylphosphate;an orally active derivative of 1-β-Darabinofuranosylcytosine.” Jpn. J.Cancer Res. 80, 679 685; Korty, M. and Engels, J. (1979) “The effects ofadenosine- and guanosine 3′,5′ phosphoric and acid benzyl esters onguinea-pig ventricular myocardium.” Naunyn-Schmiedeberg's Arch.Pharmacol. 310, 103 111; Kumar, A., Goe, P. L., Jones, A. S. Walker, R.T. Balzarini, J. and DcClereq, E. (1990) “Synthesis and biologicalevaluation of some cyclic phosphoramidate nucleoside derivatives.” J.Med. Chem, 33, 2368 2375; LeBec, C., and Huynh-Dinh, T. (1991)“Synthesis of lipophilic phosphate triester derivatives of5-fluorouridine an arabinocytidine as anticancer prodrugs.” TetrahedronLett. 32, 6553 6556; Lichtenstein, J., Barner, H. D. and Cohen, S. S.(1960) “The metabolism of exogenously supplied nucleotides byEscherichia coli.,” J. Biol. Chem. 235, 457 465; Lucthy, J., VonDaeniken, A., Friederich, J. Manthey, B., Zweifel, J., Schlatter, C. andBenn, M. H. (1981) “Synthesis and toxicological properties of threenaturally occurring cyanocpithioalkancs”. Mitt. Geg. Lebensmittelunters.Hyg. 72, 131 133 (Chem. Abstr. 95, 127093); McGigan, C. Tollerfield, S.M. and Riley, P. a. (1989) “Synthesis and biological evaluation of somephosphate triester derivatives of the anti-viral drug Ara.” NucleicAcids Res. 17, 6065 6075; McGuigan, C., Devine, K. G., O'Connor, T. J.,Galpin, S. A., Jeffries, D. J. and Kinchington, D. (1990a) “Synthesisand evaluation of some novel phosphoramidate derivatives of3′-azido-3′-deoxythymidine (AZT) as anti-HIV compounds.” Antiviral Chem.Chemother. 1 107 113; McGuigan, C., O'Connor, T. J., Nicholls. S. R.Nickson, C. and Kinchington, D. (1990b) “Synthesis and anti-HIV activityof some novel substituted dialkyl phosphate derivatives of AZT andddCyd.” Antiviral Chem. Chemother. 1, 355 360; McGuigan, C., Nicholls,S. R., O'Connor, T. J., and Kinchington, D. (1990c) “Synthesis of somenovel dialkyl phosphate derivative of 3′-modified nucleosides aspotential anti-AIDS drugs.” Antiviral Chem. Chemother. 1, 25 33;McGuigan, C., Devin, K. G., O'Connor, T. J., and Kinchington, D. (1991)“Synthesis and anti-HIV activity of some haloalkyl phosphoramidatederivatives of 3′-azido-3′-deoxythylmidine (AZT); potent activity of thetrichloroethyl methoxyalaninyl compound.” Antiviral Res. 15, 255 263;McGuigan, C., Pathirana, R. N., Balzarini, J. and DeClercq, E. (1993b)“Intracellular delivery of bioactive AZT nucleotides by aryl phosphatederivatives of AZT.” J. Med. Chem. 36, 1048 1052.

Alkyl hydrogen phosphate derivatives of the anti-HIV agent AZT may beless toxic than the parent nucleoside analogue. Antiviral Chem.Chemother. 5, 271 277; Meyer, R. B., Jr., Shuman, D. A. and Robins, R.K. (1973) “Synthesis of purine nucleoside 3′,5′-cyclicphosphoramidates.” Tetrahedron Lett. 269 272; Nagyvary, J. Gohil, R. N.,Kirchner, C. R. and Stevens, J. D. (1973) “Studies on neutral esters ofcyclic AMP,” Biochem. Biophys. Res. Commun. 55, 1072 1077; Namane, A.Gouycttc, C., Fillion, M. P., Fillion, G. and Huynh-Dinh, T. (1992)“Improved brain delivery of AZT using a glycosyl phosphotriesterprodrug.” J. Med. Chem. 35, 3039 3044; Nargeot, J. Nerbonne, J. M.Engels, J. and Leser, H. A. (1983) Natl. Acad. Sci. U.S.A. 80, 23952399; Nelson, K. A., Bentrude, W. G. Stser, W. N. and Hutchinson, J. P.(1987) “The question of chair-twist equilibria for the phosphate ringsof nucleoside cyclic 3′,5′-monophosphates. ¹HNMR and x-raycrystallographic study of the diastereomers of thymidine phenyl cyclic3′,5′-monophosphate.” J. Am. Chem. Soc. 109, 4058 4064; Nerbonne, J. M.,Richard, S., Nargeot, J. and Lester, H. A. (1984) “New photoactivatablecyclic nucleotides produce intracellular jumps in cyclic AMP and cyclicGMP concentrations.” Nature 301, 74 76; Neumann, J. M., Herv_, M.,Debouzy, J. C., Guerra, F. I., Gouyette, C., Dupraz, B. and Huyny-Dinh,T. (1989) “Synthesis and transmembrane transport studies by NMR of aglucosyl phospholipid of thymnidine.” J. Am. Chem. Soc. 111, 4270 4277;Ohno, R., Tatsumi, N., Hirano, M., Imai, K. Mizoguchi, H., Nakamura, T.,Kosaka, M., Takatuski, K., Yamaya, T., Toyama K., Yoshida, T., Masaoka,T., Hashimoto, S., Ohshima, T., Kimura, I., Yamada, K. and Kimura, J.(1991) “Treatment of myelodysplastic syndromes with orally administered1-β-D-arabinouranosylcytosine-5′ stearylphosphate.” Oncology 48, 451455. Palomino, E., Kessle, D. and Horwitz, J. P. (1989) “Adihydropyridine carrier system for sustained delivery of 2′,3′dideoxynucleosides to the brain.” J. Med. Chem. 32, 22 625; Perkins, R.M., Barney, S. Wittrock, R., Clark, P. H., Levin, R. Lambert, D. M.,Petteway, S. R., Serafinowska, H. T., Bailey, S. M., Jackson, S.,Harnden, M. R. Ashton, R., Sutton, D., Harvey, J. J. and Brown, A. G.(1993) “Activity of BRL47923 and its oral prodrug, SB203657A against arauscher murine leukemia virus infection in mice.” Antiviral Res. 20(Suppl. 1). 84; Piantadosi, C., Marasco, C. J., Jr., Norris-Natschke, S.L., Meyer. K. L., Gumus, F., Surles, J. R., Ishaq, K. S., Kucera, L. S.Iyer, N., Wallen, C. A., Piantadosi, S. and Modest, E. J. (1991)“Synthesis and evaluation of novel ether lipid nucleoside conjugates foranti-HTV-1 activity.” J. Med. Chem. 34, 1408 1414; Pompon, A., Lefebvre,I., Imbach, J. L., Kahn, S. and Farquhar, D. (1994). “Decompositionpathways of the mono- and bis(pivaloyloxymethyl) esters ofazidothymidine-5′-monophosphate in cell extract and in tissue culturemedium; an application of the ‘on-line ISRP-cleaning HPLC technique.”Antiviral Chem Chemother. 5, 91 98; Postemark, T. (1974) “Cyclic AMP andcyclic GMP.” Annu. Rev. Pharmacol. 14, 23 33; Prisbe, E. J., Martin, J.C. M., McGhee, D. P. C., Barker, M. F., Smee, D. F. Duke, A. E.,Matthews, T. R. and Verheyden, J. P. J. (1986) “Synthesis and antiherpesvirus activity of phosphate an phosphonate derivatives of9-[(1,3-dihydroxy-2-propoxy)methyl]guanine.” J. Med. Chem. 29, 671 675;Pucch, F., Gosselin, G., Lefebvre, I., Pompon, a., Aubertin, A. M. Dim,and Imbach, J. L. (1993) “Intracellular delivery of nucleosidemonophosphate through a reductase-mediated activation process.”Antiviral Res. 22, 155 174; Pugaeva, V. P., Klochkeva, S. I., Mashbits,F. D. and Eizengart, R. S. (1969). “Toxicological assessment and healthstandard ratings for ethylene sulfide in the industrial atmosphere.”Gig. Trf. Prof. Zabol. 14, 47 48 (Chem. Abstr. 72, 212); Robins, R. K.(1984) “The potential of nucleotide analogs as inhibitors of Retroviruses and tumors.” Pharm. Res. 11 18; Rosowsky, A., Kim. S. H., Rossand J. Wick, M. M. (1982) “Lipophilic 5′-(alkylphosphate) esters of1-β-D-arabinofiiranosylcytosine and its N⁴-acyl and2.2′-anhydro-3′-O-acyl derivatives as potential prodrugs.” J. Med. Chem.25, 171 178; Ross, W. (1961) “Increased sensitivity of the walkerturnout towards aromatic nitrogen mustards carrying basic side chainsfollowing glucose pretreatment.” Biochem. Pharm. 8, 235 240; Ryu, E. K.,Ross, R. J. Matsushita, T., MacCoss, M., Hong, C. I. and West, C. R.(1982). “Phospholipid-nucleoside conjugates. 3. Synthesis andpreliminary biological evaluation of 1-β-D-arabinofuranosylcytosine5′diphosphate [−], 2-diacylglycerols.” J. Med. Chem. 25, 1322 1329;Saffhill, R. and Hume, W. J. (1986) “The degradation of5-iododeoxyuridine and 5-bromoethoxyuridine by serum from differentsources and its consequences for the use of these compounds forincorporation into DNA.” Chem. Biol. Interact. 57, 347 355; Saneyoshi,M., Morozumi, M., Kodama, K., Machida, J., Kuninaka, A. and Yoshino, H.(1980) “Synthetic nucleosides and nucleotides. XVI. Synthesis andbiological evaluations of a series of 1-β-D-arabinofuranosylcytosine5′-alky or arylphosphates.” Chem Pharm. Bull. 28, 2915 2923; Sastry, J.K., Nehete, P. N., Khan, S., Nowak, B. J., Plunkett, W., Arlinghaus, R.B. and Farquhar, D. (1 992) “Membrane-permeable dideoxyuridine5′-monophosphate analogue inhibits human immunodeficiency virusinfection.” Mol. Pharmacol. 41, 441 445; Shaw, J. P., Jones, R. J.Arimilli, M. N., Louie, M. S., Lee, W. A. and Cundy, K. C. (1994) “Oralbioavailability of PMEA from PMEA prodrugs in male Sprague-Dawley rats.”9th Annual AAPS Meeting. San Diego, Calif. (Abstract). Shuto, S., Ueda,S., Imamura, S., Fukukawa, K. Matsuda, A. and Ueda, T. (1987) “A facileone-step synthesis of 5′ phosphatidiylnucleosides by an enzymatictwo-phase reaction.” Tetrahedron Lett. 28, 199 202; Shuto, S. Itoh, H.,Ucda, S., Imamura, S., Kukukawa, K., Tsujino, M., Matsuda, A. and Ucda,T. (1988) Pharm. Bull. 36, 209 217. An example of a useful phosphateprodrug group is the S-acyl-2-thioethyl group, also referred to as“SATE”.

VI. Methods of Treatment

The compositions described herein can be used to treat patients infectedwith HIV-1 and HIV-2, to prevent an infection by HIV-1 and HIV-2, or toeradicate an HIV-1 or HIV-2 infection.

When the treatment involves co-administration of the JAK inhibitorsdescribed herein and nucleoside antiviral agents and/or non-thymidinenucleoside antiviral agents, the HIV-1 or HIV-2 may already havedeveloped one or more mutations, such as the M184V, K65R mutation orTAMS. In such a case, the second agent will ideally be selected to beactive against HIV-1 or HIV-2 that has these mutations. Methods forselecting appropriate antiretroviral therapy for patients with variousmutations in their HIV-1 or HIV-2 are known to those of skill in theart.

When the treatment involves the co-administration of an adenine,cytosine, thymidine, and guanine nucleoside antiviral agent, as well asthe additional antiviral agent(s), ideally the administration is to apatient who has not yet developed any resistance to these antiviralagents or has been off therapy for at least three months. In that case,it may be possible to actually cure an infected patient if the therapycan treat substantially all of the virus, substantially everywhere itresides in the patient. However, even in the case of infection by aresistant virus, the combination therapy should be effective against allknown resistant viral strains, because there is at least one agentcapable of inhibiting such a virus in this combination therapy, andbecause the JAK inhibitors do not function in the same manner as theconventional NRTI, NNRTI, protease inhibitors, entry inhibitors,integrase inhibitors, and the like, and thus remain effective againststrains that have mutated following exposure to these agents.

The compounds can be used in different ways to treat or prevent HIV,and, in one embodiment, to cure an HIV infection. In one embodiment, acombination of a JAK inhibitor as described herein, a macrophagedepleting agent (e.g., clodronate-loaded liposomes, gadolinium chloride(GdCl)), plus HAART therapy is used. The strategy involves reducingviral loads with traditional HAART and JAK inhibitor therapy. Then,macrophages are systemically depleted (typically without discriminationfor infected versus infected macrophages). HAART and JAK inhibitortherapy would be maintained during macrophage depletion. Then, treatmentwith the macrophage depleting agent is withdrawn, while treatment withHAART and the JAK inhibitor is maintained.

In one aspect of this embodiment, HAART is then withdrawn, while JAKinhibitor therapy is maintained, optionally while monitoring viralrebound.

In another aspect of this embodiment, both HAART and JAK inhibitortherapy are then withdrawn, optionally while monitoring viral rebound.

In another embodiment, viral loads are reduced with traditionalHAART+JAK inhibitors, specifically one or both of Tofacitinib andJakafi, as described herein. Then, macrophages are systemically depleted(typically without discrimination for infected versus infectedmacrophages) with Boniva or Fosamax (both of these drugs are potentmacrophage depleting agents). HAART+JAK inhibitor therapy is maintainedduring macrophage depletion. Then, treatment with the macrophagedepleting agent is withdrawn, while treatment with HAART and the JAKinhibitor is maintained.

In one aspect of this embodiment, HAART is then withdrawn, while JAKinhibitor therapy with one or both of Tofacitinib and Jakafi ismaintained, optionally while monitoring viral rebound.

In another aspect of this embodiment, both HAART and JAK inhibitortherapy with one or both of Tofacitinib and Jakafi are then withdrawn,optionally while monitoring viral rebound.

In another embodiment, a combination of a histone deacetylase inhibitor(HDAC inhibitor) or interleukin 7 (IL-7) and HAART and a JAK inhibitoris used. One limitation associated with treating HIV is that while it isnot fully understood how HIV-1 evades the immune response andestablishes latency in resting cells, it is believed that a variety ofsignalling molecules and transcription factors appear to play a role,and thus offer potential targets for intervention. Thus, in thisembodiment, TL-7 is used to confer reactivation of resting cells,effectively flushing HIV-1 out of hiding, and histone deacetylase (HDAC)inhibitors are used to confer reactivation by up regulation of pro-HIVgenes, effectively coaxing virus out from previously resting cells. Inthis manner, latent HIV is eradicated. An example of a reactivationagent that could be used in this manner is panobinostate, which isdescribed, for example, in Lewin, et al., “HIV cure and eradication: howwill we get from the laboratory to effective clinical trials?” AIDS:24Apr. 2011. Representative HDAC inhibitors include Vorinostat, Romidepsin(trade name Istodax), Panobinostat (LBH589), Valproic acid (including Mgvalproate and other salt forms), Belinostat (PXD101), Mocetinostat(MGCD0103), PCI-24781, Entinostat (MS-275), SB939, Resminostat(4SC-201), Givinostat (ITF2357), CUDC-101, AR-42, CHR-2845, CHR-3996,4SC-202, sulforaphane, suberoylanilide hydroxamic acid (SAHA), BML-210,M344, CI-994; CI-994 (Tacedinaline); BML-210; M344; MGCD0103(Mocetinostat); and Tubastatin A. Additional HDAC inhibitors aredescribed in U.S. Pat. No. 7,399,787.

The strategy involves reducing viral loads with traditional HAART andJAK inhibitor therapy. Then, the patient is treated with a reactivationagent (as defined in Lewin et al., supra), such as panobinostat.

In one aspect of this embodiment, both HAART and JAK inhibitor therapyare maintained during reactivation, and in another aspect of thisembodiment, HAART, but not JAK inhibitor therapy, is maintained duringreactivation.

Treatment with the reactivation agent is then withdrawn, whilecontinuing treatment with HAART and one or more JAK inhibitors, such asTofacitinib and Jakafi as defined herein.

In one aspect of this embodiment, HAART is then withdrawn, while JAKinhibitor therapy is maintained, optionally while monitoring viralrebound.

In another aspect of this embodiment, both HAART and JAK inhibitortherapy are then withdrawn, optionally while monitoring viral rebound.

In another embodiment, the JAK inhibitors are administered to a patientbefore, during, or after administration of a vaccine and/or animmunostimulant. The use of immunostimulants can provide an optimalantiretroviral regimen. The immunostimulatory treatments include, butare not limited to, therapies from two functional classes: 1) agentsthat target actively replicating cells and 2) agents activating latentlyinfected cells.

In addition to the JAK inhibitors and immunomodulatory agents, HAART canalso be provided. The JAK inhibitors, optionally with co-administeredHAART, can suppress virus to undetectable or virtually undetectablelevels. The addition of an immunomodulatory therapy that specificallytargets viral reservoirs can, ideally, lead to a cure, or at leastremove virus from one or more viral reservoirs.

Immunostimulants

The term “immunostimulant” is used herein to describe a substance whichevokes, increases and/or prolongs an immune response to an antigen.While the present application distinguishes between an “antigen” and an“immunostimulant” it should be noted that this is merely for reasons ofclarity and ease of description. It should be understood that theimmunostimulant could have, and in many cases preferably has, antigenicpotential itself.

Immunomodulatory agents modulate the immune system, and, as used herein,immunostimulants are also referred to as immunomodulatory agents, whereit is understood that the desired modulation is to stimulate the immunesystem.

There are two main categories of immunostimulants, specific andnon-specific. Specific immunostimulants provide antigenic specificity inimmune response, such as vaccines or any antigen, and non-specificimmunostimulants act irrespective of antigenic specificity to augmentimmune response of other antigen or stimulate components of the immunesystem without antigenic specificity, such as adjuvants and non-specificimmunostimulators.

Examples of immunostimulants include levamisole, thalidomide, erythemanodosum leprosum, BCG, cytokines such as interleukins or interferons,including recombinant cytokines and interleukin 2 (aldeslukin), 3D-MPL,QS21, CpG ODN 7909, miltefosine, anti-PD-1 or PD-1 targeting drugs, andacid (DCA, a macrophage stimulator), imiquimod and resiquimod (whichactivate immune cells through the toll-like receptor 7), chlorooxygencompounds such as tetrachlorodecaoxide (TCDO), agonistic CD40antibodies, soluble CD40L, 4-1BB:4-1BBL agonists, OX40 agonists, TLRagonists, moieties that deplete regulatory T cells, arabinitol-ceramide,glycerol-ceramide, 6-deoxy and 6-sulfono-myo-insitolceramide, iNKTagonists, TLR agonists.

WF 10 [Immunokine, Macrokine] is a 1:10 dilution of tetrachlorodecaoxide(TCDO) formulated for intravenous injection. WF 10 specifically targetsmacrophages, and modulates disease-related up-regulation of immuneresponses in vivo.

3D-MPL is an immunostimulant derived from the lipopolysaccharide (LPS)of the Gram-negative bacterium Salmonella minnesota. MPL has beendeacylated and is lacking a phosphate group on the lipid A moiety. Thischemical treatment dramatically reduces toxicity while preserving theimmunostimulant properties (Ribi, 1986). Ribi Immunochemistry producesand supplies MPL to GSK-Biologicals.

QS21: is a natural saponin molecule extracted from the bark of the SouthAmerican tree Quillaja saponaria Molina. A purification techniquedeveloped to separate the individual saponins from the crude extracts ofthe bark, permitted the isolation of the particular saponin, QS21, whichis a triterpene glycoside demonstrating stronger adjuvant activity andlower toxicity as compared with the parent component. QS21 has beenshown to activate MHC class I restricted CTLs to several subunit Ags, aswell as to stimulate Ag specific lymphocytic proliferation (Kensil,1992). Aquila (formally Cambridge Biotech Corporation) produces andsupplies QS21 to GSK-Biologicals.

CpG ODN 7909 is a synthetic single-stranded phosphorothioateoligodeoxy-nucleotide (ODN) of 24 bases length. Its base sequence, whichis 5′-TCGTCGTTTTG-TCGTTTTGTCGT-3′, has been optimized for stimulation ofthe human immune system. CpG DNA or synthetic ODN containing CpG motifsare known to activate dendritic cells, monocytes and macrophages tosecrete TH1-like cytokines and to induce TH1 T cell responses includingthe generation of cytolytic T cells, stimulate NK cells to secrete IFNgand increase their lytic activity, they also activate B cells toproliferate (Krieg A et al. 1995 Nature 374: 546, Chu R et al. 1997 J.Exp. Med. 186: 1623). CpG 7909 is not antisense to any known sequence ofthe human genome. CpG 7909 is a proprietary adjuvant developed by andproduced on behalf of Coley Pharmaceutical Group, Inc., Mass., US.

iNKT Agonists

A subset of T cells known as iNKT (invariant natural killer T) cells aredefined by their expression of a restricted TCR repertoire, consistingof a canonical V-alpha-14-J-alpha-18 or V-alpha-24-J-alpha-18-alphachain in mice and humans respectively. iNKT cells recognize and becomeactivated in response to self or foreign antigenic lipids presented bynon-polymorphic CD1d molecules expressed on the surface of APCs. iNKTcells are activated in response to a variety of infections, and duringinflammatory and autoimmune diseases. iNKT cells provide a means oflinking and coordinating innate and adaptive immune responses, as theirstimulation can induce the downstream activation of DCs, NK cells, B andT cells. It has been demonstrated in vitro that iNKT cells stimulate Bcell proliferation and antibody production.

NKT cells can be activated by alpha-galactosyl-ceramide (alpha-GalCer)or its synthetic analog KRN 7000 (U.S. 2003/0157135). Alpha-GalCer canstimulate NK activity and cytokine production by NKT cells (U.S.2003/0157135). Alpha-GalCer and related glycosylceramides not onlyfunction as antigens, but can also be used as soluble adjuvants capableof enhancing and/or extending the duration of the protective immuneresponses induced by other antigens.

Thus, in some embodiments of the present invention the immunostimulantmay be an iNKT cell agonist. The agonist may be an exogenous orendogenous agonist. It may be a glycosidic agonist (such asalpha-galactasylceramide) or a non-glycosidic agonist (such asthreitoceramide).

Immunostimulatory Lipids or Glycolipids

In some embodiments, the immunostimulant may be a lipid or a glycolipid.Glycolipids presented by CD1 can be grouped into different classesincluding for example diacylglycerolipids, sphingolipids, mycolates andphosphomycoketides (Zajonc and Krenenberg, Current Opinion in StructuralBiology, 2007, 17:521-529). Microbial antigens from pathogenicmycobacteria, such as glucose monomycolates (GMM), mannosylphosphomycoketides and phosphatidylinositol mannosides are known to bepotent ligands for human T cells when presented by group I CD1 molecules(Zajonc an Kronenherg, supra). The immunostimulant can be aglycosylceramide, for example alpha-galactosylceramide (KRN 7000,US2003/0157135) or an analogue thereof, such as for examplethreitolceramide (IMM47) or other non-glycosidic iNKT cell agonists (asdescribed in Silk et al. Cutting Edge J. Immunol, 2008). Furtheranalogues which may be used in accordance with the invention and methodsof producing such analogues are disclosed in WO2007/050668, which isincorporated herein by reference.

TLR Agonists

Intracellular TLRs such as TLRs 3, 7, 8 and 9 recognize nucleic acids.As such, synthetic oligodeoxynucleotides (ODN) such as the TLR9 agonistCpG have previously been used as immunostimulants. These TLRimmunostimulants operate by a different mechanism than that employed bylipids such as alphaGalCer. These immunostimulants directly activate thecell that they are taken up by, culminating in, for example, thesecretion of cytokines and chemokines that result in the furtherstimulation of immune responses.

The TLR expression pattern is specific for each cell type (Chiron et al,2009). TLR expression in human B cells is characterized by highexpression of TLR 1, 6, 7, 9 and 10, with the expression pattern varyingduring B-cell differentiation.

Soluble CpG ODNs are rapidly internalized by immune cells and interactwith TLR9 that is present in endocytic vesicles. Cellular activation bymost members of the TLR family (including TLR9) involves a signalingcascade that proceeds through myeloid differentiation primary responsegene 88 (MYD88), interleukin-1 (IL-1), receptor-activated kinase (IRAK)and tumor-necrosis factor receptor (TNFR)-associated factor 6 (TRAF6),and culminates in the activation of several transcription factors,including nuclear factor-kappaB (NF-kappaB), activating protein 1 (AP1),CCAAT-enhancer binding protein (CEBP) and cAMP-responsive elementbinding protein (CREB). These transcription factors directly upregulatecytokine/chemokine gene expression. B cells and plasmacytoid dendriticcells (pDCs) are the main human cell types that express TLR9 and responddirectly to CpG stimulation. Activation of these cells by CpG DNAinitiates an immunostimulatory cascade that culminates in the indirectmaturation, differentiation and proliferation of natural killer (NK)cells, T cells and monocytes/macrophages. Together, these cells secretecytokines and chemokines that create a pro-inflammatory (IL-1, IL-6,IL-18 and TNF) and T.sub.H1-biased (interferon-.gamma., IFN-.gamma., andTL-12) immune milieu (Klinman, 2004, Nature Reviews, 4:249).

Thus, in some embodiments the immunostimulant is a TRL agonist. Forexample, it is an endosomal TLR agonist, in particular a nucleic acid,such as for example DNA, RNA (either double or single stranded). Theimmunostimulant may, for example comprise a CpG oligodeoxynucleotide ora poly-U nucleic acid.

Saponins

Saponins are taught in: Lacaille-Dubois, M and Wagner H. (1996. A reviewof the biological and pharmacological activities of saponins.Phytomedicine vol 2 pp 363-386). Saponins are steroid or triterpeneglycosides widely distributed in the plant and marine animal kingdoms.

Saponins are known as adjuvants in vaccines for systemic administration.The adjuvant and haemolytic activity of individual saponins has beenextensively studied in the art (Lacaille-Dubois and Wagner, supra). Forexample, Quil A (derived from the bark of the South American treeQuillaja Saponaria Molina), and fractions thereof, are described in U.S.Pat. No. 5,057,540 and “Saponins as vaccine adjuvants”, Kensil, C. R.,Crit Rev Ther Drug Carrier Syst, 1996, 12 (1-2):1-55; and EP 0 362 279B1. Particulate structures, termed Immune Stimulating Complexes(ISCOMS), comprising Quil A or fractions thereof, have been used in themanufacture of vaccines (Morein, B., EP 0 109 942 B1). These structureshave been reported to have adjuvant activity (EP 0 109 942 B1; WO96/11711). The hemolytic saponins QS21 and QS17 (HPLC purified fractionsof Quil A) have been described as potent systemic adjuvants, and themethod of their production is disclosed in U.S. Pat. No. 5,057,540 andEP 0 362 279 B1. Also described in these references is the use of QS7 (anon-haemolytic fraction of Quil-A) which acts as a potent adjuvant forsystemic vaccines. Use of QS21 is further described in Kensil et al.(1991. J. Immunology vol 146, 431-437). Combinations of QS21 andpolysorbate or cyclodextrin are also known (WO 99/10008). Particulateadjuvant systems comprising fractions of QuilA, such as QS21 and QS7 aredescribed in WO 96/33739 and WO 96/11711.

Other saponins which have been used in systemic vaccination studiesinclude those derived from other plant species such as Gypsophila andSaponaria (Bomford et al., Vaccine, 10(9):572-577, 1992).

Cytokines

TH-1 type cytokines, e.g., IFN-gamma, TNF-alpha, IL-2, IL-12, IL-18,etc, tend to favor the induction of cell mediated immune responses to anadministered antigen. In contrast, high levels of Th2-type cytokines(e.g., IL4, IL-5, IL-6 and IL-10) tend to favor the induction of humoralimmune responses. Interleukin-18 (IL-18), also known as interferon-gamma(IFNg) inducing factor, has been described as an pleotropic cytokinewith immunomodulatory effects that stimulates patient's own immunesystem against disease. IL-18 has several bioactivities, including theability to promote the differentiation of naive CD4 T cells into Th1cells, to stimulate natural killer (NK) cells, natural killer T (NKT)cells, and to induce the proliferation of activated T cells,predominantly cytotoxic T cells (CD8+ phenotype) to secrete gammainterferon (IFN-gamma) (Okamura H. et al. 1998, Adv. Immunol. 70:281-312). IL-18 also mediates Fas-induced tumor death, promotes theproduction of IL-1a and GMCSF, and has anti-angiogenic activity. IFN-α2a, including pegylated versions thereof (Pegasys), can also be used.Recombinant human Interleukin-7 (r-hIL-7/CYT107) can also be used.

Vaccines

As used herein, “vaccine” includes all prophylactic and therapeuticvaccines. A vaccine includes an antigen or immunogenic derivative, andan adjuvant. As used herein, the vaccines can be any vaccine thatinhibits any of the viruses described herein, including anti-HIVvaccines which inhibit HIV through any mechanism.

Where the vaccine is an anti-HIV vaccine, it ideally inhibits or stopsthe HIV virion replication cycle at any one of the following phases ofthe HIV virion cycle:

Phase I. Free State

Phase II. Attachment

Phase III. Penetration

Phase IV. Uncoating

Phase V. Replication

Phase VI. Assembling

Phase VII. Releasing

While many antiviral vaccines use live viruses, with respect to HTVvaccines, it is not advisable to use live viruses, due to the risk ofinfection. However, it is known that deletion of the HIV nef geneattenuates the virus. Desrosiers and his associates have demonstratedthat vaccination of macaques with nef-deleted SIV protected wild-typeSIV challenge (Daniels, M. D. et al. Science 258:1938 (1992);Desrosiers, R. C., et al. Proc. Natl. Acad. Sci. USA 86:6353 (1989)) andothers have demonstrated that nef gene is dispensable for SIV and HIVreplication (Daniels, M. D. et al. Science 258:1938 (1992); Gibbs, J.S., et al. AIDS Res. and Human Retroviruses 10:343 (1994); Igarashi, T.,et al. J. Gen. Virol. 78:985 (1997); Kestler III, H. W., et al. Cell65:651 (1991)). Furthermore, deletion of nef gene renders the virus tobe non-pathogenic in the normally susceptible host (Daniels, M. D. e tal. Science 258:1938 (1992)).

In terms of antigens, subunit vaccines can be used (Cooney E L, et al.,Proc Natl Acad Sci USA 1993; 90; 1882-86; McElrath M J, et al. J InfectDis. 169: 41-47 (1994); Graham B S, et al. J Infect Dis 166: 244-52(1992); and Graham B S, et al. J Infect Dis 167: 533-37 (1993)).HIV-derived antigens include HIV-1 antigen gp120, tat, nef, reversetranscriptase, gag, gp120 and gp160, and various targets in pol Oneexamples of an HIV vaccine is the DermaVir therapeutic HIV vaccine,currently in Phase II clinical studies.

The vaccines of the present invention may additionally contain suitablediluents, adjuvants and/or carriers. In some embodiments, the vaccinescontain an adjuvant which can enhance the immunogenicity of the vaccinein vivo. The adjuvant may be selected from many known adjuvants in theart, including the lipid-A portion of gram negative bacteria endotoxin,trehalose dimycolate of mycobacteria, the phospholipid lysolecithin,dimethyldictadecyl ammonium bromide (DDA), certain linearpolyoxypropylene-polyoxyethylene (POP-POE) block polymers, aluminumhydroxide, and liposomes. The vaccines may also include cytokines thatare known to enhance the immune response including GM-CSF, IL-2, IL-12,TNF-α and IFNγ.

The dose of the vaccine may vary according to factors such as thedisease state, age, sex, and weight of the individual, and the abilityof antibody to elicit a desired response in the individual. Dosageregime may be adjusted to provide the optimum therapeutic response. Forexample, several divided doses may be administered daily or the dose maybe proportionally reduced as indicated by the exigencies of thetherapeutic situation. The dose of the vaccine may also be varied toprovide optimum preventative dose response depending upon thecircumstances.

The vaccines may be administered in a convenient manner such as byinjection (subcutaneous, intravenous, intramuscular, etc.), oraladministration, inhalation, transdermal administration (such as topicalcream or ointment, etc.), or suppository applications.

Recombinant Retrovirus

The recombinant retrovirus of the present invention can be anyretrovirus, including HIV-1, HIV-2, SIV, HTLV-1. Preferably theretrovirus is a human immunodeficiency virus selected from HIV-1 andHIV-2, more preferably, the retrovirus is HIV-1.

The vaccine can be an essentially non-cytolytic retrovirus, wherein theterm “essentially non-cytolytic” means that the retrovirus does notsignificantly damage or kill the cells it infects. In one embodiment,the natural signal sequence of HIV-1 envelope glycoprotein gp120 (NSS)is modified to be essentially non-cytolytic, or is replaced with anessentially non-cytolytic signal sequence.

In one embodiment, the present invention provides an essentiallynon-cytolytic recombinant HIV-1 capable of highly efficient replicationwherein the NSS of the virus' envelope glycoprotein is modifiedsufficiently to prevent cell damage by the virus, preferably byeliminating positively charged amino acids, even more preferably, suchelimination or modification resulting in no more than one (1) andpreferably zero (0) positively charged amino acids. The positivelycharged amino acids which may be modified or replaced include lysine andarginine.

In another embodiment, replacement of the natural signal sequenceresults in a more efficient replication of HIV. Accordingly the presentinvention provides an essentially non-cytolytic recombinant HIV-1capable of highly efficient replication wherein the NSS of the virus'envelope glycoprotein is replaced with an essentially non-cytolytic andmore efficient signal sequence. In a preferred embodiment, replacementof the NSS of the envelope glycoprotein of HIV-1 with either themellitin or IL-3 signal sequence decreases the cytotoxicity of theretrovirus. As such, the present invention includes within its scopereplacement of NSS with any signal sequence which renders the retrovirusessentially non-cytolytic. The inventors have also shown thatreplacement of the NSS with mellitin or IL-3 signal sequences results ina greater level of production and secretion of gp120, in addition to thereduced cytotoxicity. The inventors have also shown that replacement ofthe NSS results in partial deletion the vpu gene. Studies have shown thevpu gene can be completely deleted without any measurable impact on thevirus' ability to replicate (James et al. AIDS Res. Human Retrovirus10:343-350, 1994).

In another embodiment, the retrovirus is rendered avirulent. In apreferred embodiment, the virus is rendered avirulent by deleting thenef gene. Accordingly, the present invention provides an avirulent,essentially non-cytolytic retrovirus which contains a sufficientdeletion of the nef gene to render the virus non-pathogenic and whereinthe virus' envelope glycoprotein gp1²⁰ coding sequence is replaced witha more efficient signal sequence. As used herein, “sufficient deletion”means deletion of enough of the sequence to prevent transcription andthereby production of the nef protein product.

In a further embodiment, the retrovirus is rendered avirulent,essentially non-cytolytic, and contains a sufficient deletion of the nefgene and the vpu gene to render the virus non-pathogenic.

Recombinant retroviruses be prepared using techniques known in the art.In one embodiment, the retrovirus can be introduced in a host cell underconditions suitable for the replication and expression of the retrovirusin the host.

The essentially non-cytolytic and avirulent retroviruses can typicallybe produced in large quantities and in a form that is non-pathogenic tothe patient. The viruses can be used, in combination with the JAKinhibitors and, optionally, with HAART, for preventing or treating aretroviral infection. In this use, an effective amount of a killedrecombinant essentially non-cytolytic avirulent retrovirus isadministered to a patient in need of treatment or prophylaxis of aretroviral infection. The term “effective amount” as used herein meansan amount effective and at dosages and for periods of time necessary toachieve the desired result.

In one embodiment, the natural signal sequence of the virus' envelopeglycoprotein, such as gp120, is modified to provide an essentiallynon-cytolytic signal sequence, and/or the virus is rendered avirulent bydeleting the nef gene. In one aspect of this embodiment, themodification to provide a non-cytolytic NSS results in no more than onepositively charged amino acid in the NSS sequence, more preferably zeropositively charged amino acids.

In another aspect of this embodiment, the natural signal sequence of thevirus' envelope glycoprotein, preferably gp120, is replaced with anessentially non-cytolytic signal sequence, and, optionally, the virus isrendered avirulent by deleting the nef gene.

In another aspect of this embodiment, where the NSS is replaced, thenon-cytolytic signal sequence is selected from the group consisting ofthe mellitin sequence and the IL-3 signal sequence.

Chimaeric Antigens

The vaccines can comprise chimaeric antigens, for example, a chimaericinfluenza-HIV vaccine. In one embodiment, the vaccine comprises theA-antigenic loop of influenza hemagglutinin (HA-A), modified to resemblethe principle neutralizing determinant (PND) of HIV envelopeglycoprotein gp120. The Chimaeric antigens can be presented as killed orattenuated virus.

Vaccine Production

To produce a vaccine, the antigen is typically combined with apharmaceutically acceptable carrier, and, typically, an adjuvant, tomake a composition comprising a vaccine. This vaccine composition isoptionally combined with an immunostimulant and administered to apatient in need of treatment or prevention of a viral infection.

In one embodiment, the vaccine includes antigens selected for more thanone virus, particularly where co-infection rates are known to be high.One example is HIV and HBV or HCV, or HIV and influenza.

A variety of adjuvants known to one of ordinary skill in the art may beadministered in conjunction with the protein in the vaccine composition.Such adjuvants include, but are not limited to the following: polymers,co-polymers such as polyoxyethylene-polyoxypropylene copolymers,including block co-polymers; polymer P1005; monotide ISA72; Freund'scomplete adjuvant (for animals); Freund's incomplete adjuvant; sorbitanmonooleate; squalene; CRL-8300 adjuvant; alum; QS 21, muramyl dipeptide;trehalose; bacterial extracts, including mycobacterial extracts;detoxified endotoxins; membrane lipids; or combinations thereof.

The vaccine formulations can be presented in unit dosage form, and canbe prepared by conventional pharmaceutical techniques. Such techniquesinclude the step of bringing into association the active ingredient andthe pharmaceutical carrier(s) or excipient(s). In general, theformulations are prepared by uniformly and intimately bringing intoassociation the active ingredient with liquid carriers. Formulationssuitable for parenteral administration include aqueous and non-aqueoussterile injection solutions which may contain anti-oxidants, buffers,bacteriostats and solutes which render the formulation isotonic with theblood of the intended recipient; and aqueous and non-aqueous sterilesuspensions which may include suspending agents and thickening agents.The formulations may be presented in unit-dose or multi-dose containers,for example, sealed ampules and vials, and may be stored in afreeze-dried (lyophilized) condition requiring only the addition of thesterile liquid carrier, for example, water for injections, immediatelyprior to use. Extemporaneous injection solutions and suspensions may beprepared from sterile powders, granules and tablets commonly used by oneof ordinary skill in the art.

Preferred unit dosage formulations are those containing a dose or unit,or an appropriate fraction thereof, of the administered ingredient. Itshould be understood that in addition to the ingredients, particularlymentioned above, the formulations of the present invention may includeother agents commonly used by one of ordinary skill in the art.

The vaccine may be administered through different routes, such as oral,including buccal and sublingual, rectal, parenteral, aerosol, nasal,intramuscular, subcutaneous, intradermal, and topical. The vaccine ofthe present invention may be administered in different forms, includingbut not limited to solutions, emulsions and suspensions, microspheres,particles, microparticles, nanoparticles, and liposomes. It is expectedthat from about 1 to 5 dosages may be required per immunization regimen.Initial injections may range from about 1 mg to 1 gram, with a preferredrange of about 10 mg to 800 mg, and a more preferred range of fromapproximately 25 mg to 500 mg. Booster injections may range from 1 mg to1 gram, with a preferred range of approximately 10 mg to 750 mg, and amore preferred range of about 50 mg to 500 mg.

The volume of administration will vary depending on the route ofadministration. Intramuscular injections may range from about 0.1 ml to1.0 ml.

The vaccines can be administered before, during or after an infection.An infected individual can receive a vaccine directed to the virusinfecting the individual, even though the levels are reduced viatreatment with the JAK inhibitors and/or HAART, stimulating the immunesystem to fight the virus that remains in the individual.

The vaccine may be stored at temperatures of from about 4 C. to −100 C.The vaccine may also be stored in a lyophilized state at differenttemperatures including room temperature. The vaccine may be sterilizedthrough conventional means known to one of ordinary skill in the art.Such means include, but are not limited to filtration, radiation andheat. The vaccine of the present invention may also be combined withbacteriostatic agents, such as thimerosal, to inhibit bacterial growth.

Treatment or Prevention of Other Viral Infections

The invention includes methods for treating or preventing, and uses forthe treatment or prophylaxis, of a Coronaviridae infection, or aFlaviviridae infection, including all members of the Hepacivirus genus(HCV), Pestivirus genus (BVDV, CSFV, BDV), or Flavivirus genus (Denguevirus, Japanese encephalitis virus group (including West Nile Virus),and Yellow Fever virus).

Viruses Characterized by the Flaviviridae Family

The Flaviviridae is a group of positive single-stranded RNA viruses witha genome size from 9-15 kb. They are enveloped viruses of approximately40-50 nm. An overview of the Flaviviridae taxonomy is available from the15 International Committee for Taxonomy of Viruses. The Flaviviridaeconsists of three genera.

Flaviviruses.

This genus includes the Dengue virus group (Dengue virus, Dengue virustype 1, Dengue virus type 2, Dengue virus type 3, Dengue virus type 4),the Japanese encephalitis virus group (Alfuy Virus, Japanese 20encephalitis virus, Kookaburra virus, Koutango virus, Kunjin virus,Murray Valley encephalitis virus, St. Louis encephalitis virus,Stratford virus, Usutu virus, West Nile Virus), the Modoc virus group,the Rio Bravo virus group (Apoi virus, Rio Brovo virus, Saboya virus),the Ntaya virus group, the Tick-Borne encephalitis group (tick bornencephalitis virus), the Tyuleniy virus 25 group, Uganda S virus groupand the Yellow Fever virus group. Apart from these major groups, thereare some additional Flaviviruses that are unclassified.

Pestiviruses.

This genus includes Bovine Viral Diarrhea Virus-2 (BVDV-2), Pestivirustype 1 (including BVDV), Pestivirus type 2 (including 30 Hog CholeraVirus) and Pestivirus type 3 (including Border Disease Virus).

Hepaciviruses.

This genus contains only one species, the Hepatitis C virus (HCV), whichis composed of many clades, types and subtypes.

Chikungunya virus, an RNA virus of the genus Alphavirus, can also betreated using the compounds described herein.

Those of skill in the art can effectively follow the administration ofthese therapies, and the development of side effects and/or resistantviral strains, without undue experimentation.

The present invention will be better understood with reference to thefollowing non-limiting examples.

Example 1: Comparison of JAK Inhibitors to Conventional AntiretroviralTherapy

Current first line highly active antiretroviral therapy (HAART) for thetreatment of human immunodeficiency virus (HIV-1) infections combinestwo nucleoside reverse transcriptase inhibitors (NRTI) together witheither a protease inhibitor (PI) or non-nucleoside reverse transcriptaseinhibitor (NNRTI). These drug combinations have markedly decreasedmortality and morbidity from HIV-1 infections in the developed world.

Existing therapies cannot eradicate HIV-1 infection because of thecompartmentalization of the virus and its latent properties. Therefore,chronic therapy remains the standard of care for the foreseeable future.Although HAART regimens are selected in part to minimize crossresistance, and thereby delay the emergence of resistant viruses, allregimens eventually fail, due primarily to lack of adherence to strictregimens, delayed toxicities and/or the emergence of drug-resistantHIV-1 strains, making it a major imperative to develop regimens thatdelay, prevent or attenuate the onset of resistance for second linetreatments for infected individuals who have already demonstratedmutations. The occurrence of common resistance mutations, includingthymidine analog mutations (TAM), K65R and M184V, need to be a continuedfocus in the rational design of HIV-1 NRTI drug development.

The objectives of this study were to evaluate JAK inhibitors that do notappear to function in the same manner as NRTI, NNRTI, proteaseinhibitors, entry inhibitors, integrase inhibitors, and the like. In thedata shown in this example, the JAK inhibitors that were evaluated wereJakafi (Incyte) and Tofacitinib (Pfizer).

PBM Cell and Mφ Protocol for Antiviral Potency

Macrophages were isolated as follows: Monocytes were isolated from buffycoats of HTV-1 negative, HBV/HCV-negative donors with density gradientcentrifugation coupled with enrichment for CD14+ monocytes with RosetteSep antibody cocktail (Stem Cell Technologies, Vancouver, BritishColumbia). Cells were seeded at a concentration of 1.0×10⁶ cells/wellfor 1 hr at 37° C. and 5% CO₂ to confer plastic adherence prior torepeated washes with 1×PBS. Macrophages were maintained in mediumcontaining 100 U/ml macrophage colony-stimulating factor (m-CSF, R&DSystems, Minneapolis, Minn.), supplemented with 20% fetal calf serum(Atlanta Biologicals, Lawrenceville, Ga.) and 1% penicillin/streptomycin(Invitrogen, Carlsbad, Calif.) for 7 days (37° C., 5% CO2) prior totesting.

Macrophage infections: Macrophages were cultured as described above for7 days. For acute infection, macrophages were serum starved for 8 hrsprior to infection and cultured for 2 hr in medium containing variousconcentrations of AZT (positive control) or Tofacitinib and Jakafi for 2hr prior to removal of drug-containing medium and 4 hr infection withHIV-1BaL at 0.1 MOI in the absence of drug. 4 hrs after infection, viruswas removed and drug-containing medium was returned to the cultures.Supernatants were collected on day 7 post-infection and HIV-1 p24 wasquantified via ELISA (Zeptometrix Corporation, Buffalo, N.Y.). EC50analysis was performed using CalcuSyn software (BioSoft Corporation,Cambridge, UK).

PBM cells were isolated as follows: Lymphocytes were isolated from buffycoats derived from healthy donors obtained from Life South Laboratories(Dunwoody, Ga.). Activated lymphocytes were maintained for 72 hrs inmedium that was supplemented with 6 pg/ml phytohemagglutinin (PHA) (CapeCod associates, East Falmouth, Mass.). Media was comprised of RPMI mediasupplemented with 20% fetal calf serum, 1% penicillin/streptomyocin and2% L-glutamine (Sigma Aldrich, San Jose, Calif.).

PBM cell infections: Testing was performed in duplicate with at least 3independent assays. Cells were incubated in RPMI medium (HyClone, Logan,Utah) containing HR-TL2 (26.5 units/ml) and 20% fetal calf serum.infections were performed by adding HIV-1_(LAI) followed by a furtherincubation at 37° C., 5% CO2, 1 hr prior to addition of drugs. Assayswere performed in 24 well plates (BD Biosciences, Franklin Lakes, N.J.).One ml of supernatant was collected after 5 days in culture and thencentrifuged at 12,000 rpm for 2 hr at 4° C. in a Jouan Br43i (ThermoElectron Corp., Marietta, Ohio). The product of the RT assay wasquantified using a Packard harvester and direct beta counter and thedata were analyzed as previously described (Schinazi et al, 1990).

Cytotoxicity Assay

The toxicity of the compounds was assessed in Vero, human PBM, CEM(human lymphoblastoid), as described previously (see Schinazi R. F.,Sommadossi J.-P., Saalmann V., Cannon D. L., Xie M.-Y., Hart G. C.,Smith G. A. & Hahn E. F. Antimicrob. Agents Chemother. 1990, 34,1061-67), and also in MØ cells. Cycloheximide was included as positivecytotoxic control, and untreated cells exposed to cell culture mediumwere included as negative controls.

The cytotoxicity IC₅₀ was obtained from the concentration-response curveusing the median effective method described previously (see Chou T.-C. &Talalay P. Adv. Enzyme Regul. 1984, 22, 27-55; Belen'kii M. S. &Schinazi R. F. Antiviral Res. 1994, 25, 1-11).

The potency and toxicity of JAK inhibitors Tofacitinib and Jakafi versusFDA approved controls AZT and 3TC was evaluated in acutely infectedactivated MØ, as well as in PBM cells. The EC₅₀ data (μM) is shown inFIG. 1. Also shown in FIG. 1 are the IC₅₀ values (μM) for thesecompounds in PBM, MØ cells, CEM cells, and Vero cells.

The data show a very large therapeutic window (ratio oftoxicity/potency), and that the JAK inhibitor compounds havesubstantially the same EC₅₀ and substantially lower IC₅₀ values as AZTand 3TC.

Cell proliferation was evaluated in activated PBM cells incubated for 5days with various concentrations of Tofacitinib and Jakafi, withcycloheximide as a positive control, and a “cells plus media” controlused as well. The data is shown in FIG. 2, in terms of total cell number(10⁶ cells) versus μM drug in medium. The data shows that Tofacitiniband Jakafi do not affect total cell proliferation at antiviralconcentrations.

Cell viability was evaluated in activated PBM cells incubated for 5 dayswith various concentrations of Tofacitinib and Jakafi, withcycloheximide as a positive control, and a “cells plus media” controlused as well. The data is shown in FIG. 3, in terms of cell viability(%) versus μM drug in medium. The data shows that Tofacitinib and Jakafido not affect total cell viability at antiviral concentrations.

Conclusion

In conclusion, Tofacitinib and Jakafi are potent, sub-micromolarinhibitors of HIV-1 replication in both PBM cells and MØ cells. Thecompounds do not affect viability or proliferation for PBM cells and MØcells, or total cell number, up to around 10 μM (2-3 logs above EC₅₀).The therapeutic window (ratio of toxicity:potency) is wide for both celltypes (24->100).

Example 2: Mitochondrial Toxicity Assays in HepG2 Cells

i) Effect of the JAK Inhibitors Described Herein on Cell Growth andLactic Acid Production:

The effect on the growth of HepG2 cells can be determined by incubatingcells in the presence of 0 μM, 0.1 μM, 1 μM, 10 μM and 100 μM drug.Cells (5×10⁴ per well) were plated into 12-well cell culture clusters inminimum essential medium with nonessential amino acids supplemented with10% fetal bovine serum, 1% sodium pyruvate, and 1%penicillin/streptomycin and incubated for 4 days at 37° C. At the end ofthe incubation period the cell number was determined using ahemocytometer. Also taught by Pan-Zhou X-R, Cui L, Zhou X-J, SommadossiJ-P, Darley-Usmer V M. “Differential effects of antiretroviralnucleoside analogs on mitochondrial function in HepG2 cells” Antimicrob.Agents Chemother. 2000; 44: 496-503. To measure the effects of thecompounds on lactic acid production. HepG2 cells from a stock culturecan be diluted and plated in 12-well culture plates at 2.5×10⁴ cells perwell. Various concentrations (0 μM, 0.1 μM, 1 μM, 10 μM and 100 μM) ofthe compounds can be added, and the cultures incubated at 37° C. in ahumidified 5% CO₂ atmosphere for 4 days. At day 4 the number of cells ineach well can be determined and the culture medium collected. Theculture medium was filtered, and the lactic acid content in the mediumdetermined using a colorimetric lactic acid assay (Sigma-Aldrich). Sincelactic acid product can be considered a marker for impairedmitochondrial function, elevated levels of lactic acid productiondetected in cells grown in the presence of the compounds would indicatea drug-induced cytotoxic effect.

ii) Effect on the Compounds on Mitochondrial DNA Synthesis:

a real-time PCR assay to accurately quantify mitochondrial DNA contenthas been developed (see Stuyver L J, Lostia S, Adams M, Mathew J S, PaiB S, Grier J, Tharnish P M, Choi Y, Chong Y, Choo H, Chu C K, Otto M J,Schinazi R F. Antiviral activities and cellular toxicities of modified2′,3′-dideoxy-2′,3′-didehydrocytidine analogs. Antimicrob. AgentsChemother. 2002; 46: 3854-60). This assay can be used to determine theeffect of the compounds on mitochondrial DNA content. In this assay,low-passage-number HepG2 cells are seeded at 5,000 cells/well incollagen-coated 96-well plates. The compounds are added to the medium toobtain final concentrations of 0 μM, 0.1 μM, 10 μM and 100 μM. Onculture day 7, cellular nucleic acids are prepared by using commerciallyavailable columns (RNeasy 96 kit; Qiagen). These kits co-purify RNA andDNA, and hence, total nucleic acids were eluted from the columns. Themitochondrial cytochrome c oxidase subunit II (COXII) gene and theB-actin or rRNA gene were amplified from 5 μl of the eluted nucleicacids using a multiplex Q-PCR protocol with suitable primers and probesfor both target and reference amplifications. For COXII the followingsense, probe and antisense primers are used, respectively:5′-TGCCCGCCATCATCCTA-3′ (SEQ ID No. 1),5′-tetrachloro-6-carboxyfluorescein-TCCTCATCGCCCTCCCATCCC-TAMRA-3′ (SEQID No. 2), and 5′-CGTCTGTTATGTAAAGGATGCGT-3′ (SEQ ID No. 3). For exon 3of the B-actin gene (GenBank accession number E01094) the sense, probe,and antisense primers are 5′-GCGCGGCTACAGCTTCA-3′ (SEQ ID No. 4),5′-6-FAMCACCACGGCCGAGCGGGATAMRA-3′ (SEQ ID No. 5), and5′-TCTCCTTAATGTCACGCACGAT-3′ (SEQ ID No. 6), respectively. The primersand probes for the rRNA gene are commercially available from AppliedBiosystems. Since equal amplification efficiencies are obtained for allgenes, the comparative CT method can be used to investigate potentialinhibition of mitochondrial DNA synthesis. The comparative CT methoduses arithmetic formulas in which the amount of target (COXII gene) isnormalized to the amount of an endogenous reference (the ß-actin or rRNAgene) and is relative to a calibrator (a control with no drug at day 7).The arithmetic formula for this approach is given by 2-ΔΔCT, where ΔΔCTis (CT for average target test sample—CT for target control)—(CT foraverage reference test-CT for reference control) (see Johnson M R, KWang, J B Smith, M J Heslin, R B Diasio. Quantitation ofdihydropyrimidine dehydrogenase expression by real-time reversetranscription polymerase chain reaction. Anal. Biochem. 2000;278:175-184). A decrease in mitochondrial DNA content in cells grown inthe presence of drug would indicate mitochondrial toxicity.

iii) Electron Microscopic Morphologic Evaluation:

NRTI induced toxicity has been shown to cause morphological changes inmitochondria (e.g., loss of cristae, matrix dissolution and swelling,and lipid droplet formation) that can be observed with ultrastructuralanalysis using transmission electron microscopy (see Cui L, Schinazi RF, Gosselin G, Imbach J L. Chu C K, Rando R F, Revankar G R, SommadossiJP. Effect of enantiomeric and racemic nucleoside analogs onmitochondrial functions in HepG2 cells. Biochem. Pharmacol. 1996, 52,1577-1584; Lewis W, Levine E S, Griniuviene B, Tankersley K O, ColacinoJ M, Sommadossi J P, Watanabe K A, Perrino F W. Fialuridinc and itsmetabolites inhibit DNA polymerase gamma at sites of multiple adjacentanalog incorporation, decrease mtDNA abundance, and cause mitochondrialstructural defects in cultured hepatoblasts. Proc Natl Acad Sci USA.1996; 93: 3592-7; Pan-Zhou X R, L Cui, X J Zhou, J P Sommadossi, V MDarley-Usmar. Differential effects of antiretroviral nucleoside analogson mitochondrial function in HepG2 cells. Antimicrob. Agents Chemother.2000, 44, 496-503). For example, electron micrographs of HepG2 cellsincubated with 10 μM fialuridine (FIAU;1,2′-deoxy-2′-fluoro-1-D-arabinofuranosly-5-iodo-uracil) showed thepresence of enlarged mitochondria with morphological changes consistentwith mitochondrial dysfunction. To determine if the JAK inhibitorcompounds promote morphological changes in mitochondria, HepG2 cells(2.5×10⁴ cells/mL) can be seeded into tissue cultures dishes (35 by 10mm) in the presence of 0 μM, 0.1 μM, 1 μM, 10 μM and 100 μM nucleosideanalog. At day 8, the cells can be fixed, dehydrated, and embedded inEponas described previously. Thin sections can be prepared, stained withuranyl acetate and lead citrate, and then examined using transmissionelectron microscopy.

Example 3: Mitochondrial Toxicity Assays in Neuro2A Cells

To estimate the potential of the JAK inhibitor compounds to causeneuronal toxicity, mouse Neuro2A cells (American Type Culture Collection131) can be used as a model system (see Ray A S, Hernandez-Santiago B I,Mathew J S, Murakami E, Bozeman C, Xie M Y, Dutschman G E, Gullen E,Yang Z, Hurwitz S, Cheng Y C, Chu C K, McClure H, Schinazi R F, AndersonK S. Mechanism of anti-human immunodeficiency virus activity ofbeta-D-6-cyclopropylamino-2′,3′-didehydro-2′,3′-dideoxyguanosine.Antimicrob. Agents Chemother. 2005, 49, 1994-2001). The concentrationsnecessary to inhibit cell growth by 50% (CC₅₀) can be measured using the3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide dye-basedassay, as described. Perturbations in cellular lactic acid andmitochondrial DNA levels at defined concentrations of drug can becarried out as described above.

Example 4: Assay for Bone Marrow Cytotoxicity

Primary human bone marrow mononuclear cells were obtained commerciallyfrom Cambrex Bioscience (Walkersville, Md.). CFU-GM assays can becarried out using a bilayer soft agar in the presence of 50 units/mLhuman recombinant granulocyte/macrophage colony-stimulating factor,while BFU-E assays used a methylccllulosc matrix containing 1 unit/mLcrythropoictin (sec Sommadossi J P, Carlisle R. Toxicity of3′-azido-3′-deoxythymidine and 9-(1,3-dihydroxy-2-propoxymethyl) guaninefor normal human hepatopoietic progenitor cells in vitro. Antimicrob.Agents Chemother. 1987; 31: 452-454; Sommadossi, J P, Schinazi, R F,Chu, C K, and Xie, M Y. Comparison of Cytotoxicity of the (−) and (+)enantiomer of 2′,3′-dideoxy-3′-thiacytidine in normal human bone marrowprogenitor cells. Biochem. Pharmacol. 1992; 44:1921-1925). Eachexperiment can be performed in duplicate in cells from three differentdonors. AZT can be used as a positive control. Cells can be incubated inthe presence of a JAK inhibitor compound for 14-18 days at 37° C. with5% CO₂, and colonics of greater than 50 cells can be counted using aninverted microscope to determine IC₅₀. The 50% inhibitory concentration(IC₅₀) can be obtained by least-squares linear regression analysis ofthe logarithm of drug concentration versus BFU-E survival fractions.Statistical analysis can be performed with Student's t test forindependent non-paired samples.

Example 5: Anti-HBV Assay

The anti-HBV activity of the JAK inhibitor compounds can be determinedby treating the AD-38 cell line carrying wild type HBV under the controlof tetracycline (see Ladner S. K., Otto M. J., Barker C. S., Zaifert K.,Wang G. H., Guo J. T., Seeger C. & King R. W. Antimicrob. AgentsChemother. 1997, 41, 1715-20). Removal of tetracycline from the medium[Tet (−)] results in the production of HBV. The levels of HBV in theculture supernatant fluids from cells treated with the compounds can becompared with that of the untreated controls. Control cultures withtetracycline [Tet (+)] can also be maintained to determine the basallevels of HBV expression. 3TC can be included as positive control.

Example 6: Cytotoxicity Assay

The toxicity of the compounds can be assessed in Vero, human PBM, CEM(human lymphoblastoid), MT-2, and HepG2 cells, as described previously(see Schinazi R. F., Sommadossi J.-P., Saalmann V., Cannon D. L., XieM.-Y., Hart G. C., Smith G. A. & Hahn E. F. Antimicrob. AgentsChemother. 1990, 34, 1061-67). Cycloheximide can be included as positivecytotoxic control, and untreated cells exposed to solvent can beincluded as negative controls. The cytotoxicity IC50 can be obtainedfrom the concentration-response curve using the median effective methoddescribed previously (see Chou T.-C. & Talalay P. Adv. Enzyme Regul.1984, 22, 27-55; Belen'kii M. S. & Schinazi R. F. Antiviral Res. 1994,25, 1-11).

Example 7: HCV Replicon Assay¹

Huh 7 Clone B cells containing HCV Replicon RNA can be seeded in a96-well plate at 5000 cells/well, and the JAK inhibitor compounds testedat 10 μM in 15 triplicate immediately after seeding. Following five daysincubation (37° C., 5% CO₂), total cellular RNA can be isolated by usingversaGene RNA purification kit from Gentra. Replicon RNA and an internalcontrol (TaqMan rRNA control reagents, Applied Biosystems) can beamplified in a single step multiplex Real Time RT-PCR Assay. Theantiviral effectiveness of the compounds can be calculated bysubtracting the threshold RT-PCR cycle of the test compound from thethreshold RT-PCR cycle of the no-drug control (ΔCt HCV). A ΔCt of 3.3equals a 1-log reduction (equal to 90% less starting material) inReplicon RNA levels. The cytotoxicity of the compounds can also becalculated by using the ΔCt rRNA values. (2′-Me-C) can be used as thecontrol. To determine EC₉₀ and IC₅₀ values², ΔCt: values can first beconverted into fraction of starting material³ and then were used tocalculate the % inhibition.

REFERENCES

-   1. Stuyver L et al., Ribonucleoside analogue that blocks replication    or bovine viral diarrhea and hepatitis C viruses in culture.    Antimicrob. Agents Chemother. 2003, 47, 244-254.-   2. Reed I J & Muench H, A simple method or estimating fifty percent    endpoints. Am. J. Hyg. 27: 497, 1938.-   3. Applied Biosystems Handbook

Example 8: Assay for Effectiveness Against West Nile Virus

West Nile virus drug susceptibility assays can also be done aspreviously described in: Song, G. Y., Paul, V., Choo, H., Morrey, J.,Sidwell, R. W., Schinazi, R. F., Chu, C. K. Enantiomeric synthesis of D-and L-cyclopentenyl nucleosides and their antiviral activity against HIVand West Nile virus. J. Med. Chem. 2001, 44, 3985-3993.

Example 9: Assay for Effectiveness Against Yellow Fever

Yellow fever drug susceptibility assays can also be done as previouslydescribed in: Julander, J. G., Furuta, Y., Shafer, K., Sidwell, R. W.Activity of T-1106 in a Hamster Model of Yellow Fever Virus Infection.Antimirob. Agents Chemother. 2007, 51, 1962-1966.

Example 10: Assay for Effectiveness Against Dengue

One representative high throughput assay for identifying compoundsuseful for treating Dengue is described in Lim et al., A scintillationproximity assay for dengue virus NS5 2′-O-methyltransferase-kinetic andinhibition analyses, Antiviral Research, Volume 80, Issue 3, December2008, Pages 360-369.

Dengue virus (DENV) NS5 possesses methyltransferase (MTase) activity atits N-terminal amino acid sequence and is responsible for formation of atype 1 cap structure, m7GpppAm2′-O in the viral genomic RNA. Optimal invitro conditions for DENV2 2′-O-MTase activity can be characterizedusing purified recombinant protein and a short biotinylated GTP-cappedRNA template. Steady-state kinetics parameters derived from initialvelocities can be used to establish a robust scintillation proximityassay for compound testing. Pre-incubation studies by Lim et al.,Antiviral Research, Volume 80, Issue 3, December 2008, Pages 360-369,showed that MTase-AdoMet and MTase-RNA complexes were equallycatalytically competent and the enzyme supports a random bi hi kineticmechanism. Lim validated the assay with competitive inhibitory agents,S-adenosyl-homocysteine and two homologues, sinefungin anddehydrosinefungin. A GTP-binding pocket present at the N-terminal ofDENV2 MTase was previously postulated to be the cap-binding site. Thisassay allows rapid and highly sensitive detection of 2′-O-MTase activityand can be readily adapted for high-throughput screening for inhibitorycompounds. It is suitable for determination of enzymatic activities of awide variety of RNA capping MTases.

Example 11. Anti-Norovirus Activity

Compounds can exhibit anti-norovirus activity by inhibiting noroviruspolymerase and/or helicase, by inhibiting other enzymes needed in thereplication cycle, or by other pathways.

There is currently no approved pharmaceutical treatment for Norovirusinfection (http://www.cdc.gov/ncidod/dvrd/revb/gastro/norovirus-qa.htm),and this has probably at least in part been due to the lack ofavailability of a cell culture system. Recently, a replicon system hasbeen developed for the original Norwalk G-I strain (Chang, K. O., et al.(2006) Virology 353:463-473)

Both Norovirus replicons and Hepatitis C replicons require viralhelicase, protease, and polymerase to be functional in order forreplication of the replicon to occur. Most recently, an in vitro cellculture infectivity assay has been reported utilizing Norovirusgenogroup I and II inoculums (Straub, T. M. et al. (2007) Emerg. Infect.Dis. 13(3):396-403). This assay is performed in a rotating-wallbioreactor utilizing small intestinal epithelial cells on microcarrierbeads. The infectivity assay may be useful for screening entryinhibitors.

Example 12: Bioavailability Assay in Cynomolgus Monkeys

The following procedure can be used to determine whether the compoundsare bioavailable. Within 1 week prior to the study initiation, acynomolgus monkey can be surgically implanted with a chronic venouscatheter and subcutaneous venous access port (VAP) to facilitate bloodcollection and can undergo a physical examination including hematologyand serum chemistry evaluations and the body weight recording. Eachmonkey (six total) receives approximately 250 μCi of ³H activity witheach dose of active compound at a dose level of 10 mg/kg at a doseconcentration of 5 mg/mL, either via an intravenous bolus (3 monkeys,IV), or via oral gavage (3 monkeys, PO). Each dosing syringe is weighedbefore dosing to gravimetrically determine the quantity of formulationadministered. Urine samples are collected via pan catch at thedesignated intervals (approximately 18-0 hours pre-dose, 0-4, 4-8 and8-12 hours post-dosage) and processed. Blood samples are collected aswell (pre-dose, 0.25, 0.5, 1, 2, 3, 6, 8, 12 and 24 hours post-dosage)via the chronic venous catheter and VAP or from a peripheral vessel ifthe chronic venous catheter procedure should not be possible. The bloodand urine samples are analyzed for the maximum concentration (Cmax),time when the maximum concentration is achieved (TmaX), area under thecurve (AUC), half life of the dosage concentration (TV), clearance (CL),steady state volume and distribution (Vss) and bioavailability (F).

Example 13: Cell Protection Assay (CPA)

The assay can be performed essentially as described by Baginski, S. G.;Pevear, D. C.; Seipel, M.; Sun, S. C. C.; Benetatos, C. A.; Chunduru, S.K.; Rice, C. M. and M. S. Collett “Mechanism of action of a pestivirusantiviral compound” PNAS USA 2000, 97 (14), 7981-7986. MDBK cells (ATCC)are seeded onto 96-well culture plates (4,000 cells per well) 24 hoursbefore use. After infection with BVDV (strain NADL, ATCC) at amultiplicity of infection (MOI) of 0.02 plaque forming units (PFU) percell, serial dilutions of test compounds are added to both infected anduninfected cells in a final concentration of 0.5% DMSO in growth medium.Each dilution is tested in quadruplicate.

Cell densities and virus inocula are adjusted to ensure continuous cellgrowth throughout the experiment and to achieve more than 90%virus-induced cell destruction in the untreated controls after four dayspost-infection. After four days, plates are fixed with 50% TCA andstained with sulforhodamine B. The optical density of the wells is readin a microplate reader at 550 nm.

The 50% effective concentration (EC₅₀) values are defined as thecompound concentration that achieved 50% reduction of cytopathic effectof the virus.

Example 14: Plaque Reduction Assay

For a compound, the effective concentration is determined in duplicate24-well plates by plaque reduction assays. Cell monolayers are infectedwith 100 PFU/well of virus. Then, serial dilutions of test compounds inMEM supplemented with 2% inactivated serum and 0.75% of methyl celluloseare added to the monolayers. Cultures are further incubated at 37° C.for 3 days, then fixed with 50% ethanol and 0.8% Crystal Violet, washedand air-dried. Then plaques are counted to determine the concentrationto obtain 90% virus suppression.

Example 15: Yield Reduction Assay

For a compound, the concentration to obtain a 6-log reduction in viralload is determined in duplicate 24-well plates by yield reductionassays. The assay is performed as described by Baginski, S. G.; Pevear,D. C.; Seipel, M.; Sun, S. C. C.; Benetatos, C. A.; Chunduru, S. K.;Rice, C. M. and M. S. Collett “Mechanism of action of a pestivirusantiviral compound” PNAS USA 2000, 97 (14), 7981-7986, with minormodifications.

Briefly, MDBK cells are seeded onto 24-well plates (2×10⁵ cells perwell) 24 hours before infection with BVDV (NADL strain) at amultiplicity of infection (MOI) of 0.1 PFU per cell. Serial dilutions oftest compounds are added to cells in a final concentration of 0.5% DMSOin growth medium. Each dilution is tested in triplicate. After threedays, cell cultures (cell monolayers and supernatants) are lysed bythree freeze-thaw cycles, and virus yield is quantified by plaque assay.Briefly, MDBK cells are seeded onto 6-well plates (5×10⁵ cells per well)24 h before use. Cells are inoculated with 0.2 mL of test lysates for 1hour, washed and overlaid with 0.5% agarose in growth medium. After 3days, cell monolayers are fixed with 3.5% formaldehyde and stained with1% crystal violet (w/v in 50% ethanol) to visualize plaques. The plaquesare counted to determine the concentration to obtain a 6-log reductionin viral load.

Example 16: Diagnosis of Norovirus Infection

One can diagnose a norovirus infection by detecting viral RNA in thestools of affected persons, using reverse transcription-polymerase chainreaction (RT-PCR) assays. The virus can be identified from stoolspecimens taken within 48 to 72 hours after onset of symptoms, althoughone can obtain satisfactory results using RT-PCR on samples taken aslong as 7 days after the onset of symptoms. Other diagnostic methodsinclude electron microscopy and serologic assays for a rise in titer inpaired sera collected at least three weeks apart. There are alsocommercial enzyme-linked immunoassays available, but these tend to haverelatively low sensitivity, limiting their use to diagnosis of theetiology of outbreaks. Clinical diagnosis of norovirus infection isoften used, particularly when other causative agents of gastroenteritishave been ruled out.

Example 17: In Vitro Antiviral Activity

In vitro anti-viral activity can be evaluated in the following celllines:

The Norwalk G-I strain (Chang, K. O., et al. (2006) Virology353:463-473), the GII-4 strain replicon, as well other Norovirusreplicons can be used in assays to determine the in vitro antiviralactivity of the compounds described herein, or other compounds orcompound libraries.

In some embodiments, the replicon systems are subgenomic and thereforeallow evaluation of small molecule inhibitors of non-structuralproteins. This can provide the same benefits to Norovirus drug discoverythat Hepatitis C replicons contributed to the discovery of therapeuticsuseful for treatment of that virus (Stuyver, L. J., et al. (2006)Antimicrob. Agents Chemother. 47:244-254). Both Norovirus replicons andHepatitis C replicons require viral helicase, protease, and polymeraseto be functional in order for replication of the replicon to occur. Itis believed that the compounds described herein inhibit viral polymeraseand/or viral helicase.

The in vitro cell culture infectivity assay reported using Norovirusgenogroup I and II inoculums (Straub, T. M. et al. (2007) Emerg. Infect.Dis. 13(3):396-403) can also be used. This assay can be performed in arotating-wall bioreactor utilizing small intestinal epithelial cells onmicrocarrier beads. The infectivity assay can be used for evaluatingcompounds for their ability to inhibit the desired virus.

Example 18: Antiviral Potency and Toxicity of Jakafi and Tofacitinib inPrimary Human Lymphocytes and Macrophages

The antiviral potency and toxicity of jakafi and tofacitinib wasevaluated in primary human lymphocytes and macrophages, using themethodology outlined above.

The antiviral potency against HIV-1LAI in primary human lymphocytes was0.1-0.8 μM (EC₅₀) and 4.7-15.1 μM (EC₉₀). Antiviral potency againstHIV-2 in primary human lymphocytes was 0.02-0.07 μM (EC₅₀) and 0.4-1.8μM (EC₉₀). Antiviral potency against HIV-1BaL in primary humanmacrophages was approximately 0.3 μM (EC₅₀) and 3.0 μM (EC₉₀). AZT(control) demonstrated antiviral potency against HIV-1LAI, HIV-2, andHIV-1BaL as expected. Toxicity (IC₅₀) measured with the MTT assay rangedfrom 1.3 to >100 μM depending on the cell type tested. Propidium Iodide(primary human lymphocytes) demonstrated IC₅₀>50 μM. Data are mean andstandard deviations from at least three independent experiments.

The data is shown below in Tables 1 and 2.

TABLE 1 Anti-HIV-1 Anti-HIV-1 Anti-HIV-2 Anti-HIV-2 Anti-HIV-1Anti-HIV-1 EC₅₀ in EC₉₀ in EC₅₀ in EC₉₀ in EC₅₀ in EC₉₀ in acutelyinfected acutely infected acutely infected acutely infected acutelyinfected acutely infected Compound PBM cells (μM) PBM cells (μM) PBMcells (μM) PBM cells (μM) MΦ (μM) MΦ (μM) Jakafi 0.1 ± 0.02  4.7 ± 0.070.02 ± 0.01  0.4 ± 0.2 0.3 ± 0.1 3.1 ± 1.8 Tofacitinib 0.8 ± 0.3  17.1 ±15.1 0.07 ± 0.006 1.8 ± 1.1  0.2 ± 0.08 2.9 ± 1.4 AZT 0.02 ± 0.008 0.13± 0.03 0.001 ± 0.0008 0.01 ± 0.01 0.01 ± 0.02 0.07 ± 0.12

TABLE 2 IC₅₀ in IC₅₀ in IC₅₀ in PHA + IL-2 PHA stimulated PBM cells(μM)IC₅₀ in IC₅₀ in IC₅₀ in PBM cells(μM) PBM cells(μM) *Propidium Mϕ(μM)CEM cells (μM) Vero cells(μM) Compound *MTT Assay *MTT Assay Iodideassay *MTT Assay *MTT Assay *MTT Assay Jakafi 3.1 ± 1.7 9.1 ±1.3 >50 >100 11.8 ± 1.1 29.3 ± 3.7 Tofacitinib 1.3 ± 0.9 6.3 ± 1.8 >5049.2 >100 >100 AZT >100 >100 >50 >100 14.3 56.0

Example 19: Therapeutic Index for Jakafi and Tofacitinib in PrimaryHuman Lymphocytes and Macrophages

The therapeutic index (ratio of toxicity:potency) for Jakafi andTofacitinib was evaluated in primary human lymphocytes and macrophagesusing the methodology described above. The therapeutic index ranged from1.0-31.0 for HIV-1 infection in primary human lymphocytes when using MTTassay toxicity values, and were >100 using propidium iodide toxicityvalues. The therapeutic Index ranged from 18 to >100 for HIV-2 infectionin primary human lymphocytes when using MT assay toxicity values, andwere >100 using propidium iodide toxicity values. The therapeutic indexfor HIV-1 infection in primary human macrophages was 100 (MTT assaytoxicity values).

The data are shown below in Tables 3 and 4.

TABLE 3 TI forantiviral potency TI forantiviral potency against acuteHIV-1 infection against acute HIV-1 infection TI for antiviral potencyin PBM cells versus toxicity in PBM cells versus toxicity against acuteHIV-1 infection * Calculated using MTT assay * Calculated using MTTassay in PBM cells versus toxicity toxicity values for PHA + IL-2toxicity values for PHA * Calculated using Propidium Compound stimulatedPBM cells stimulated PBM cells Iodide toxicity values Jakafi31.0 >100 >100 Tofacitinib 1.0 8.0 >100 AZT >100 >100 >100

TABLE 4 TI for antiviral potency TI for antiviral potency TI forantiviral potency against acute HIV-2 infection against acute HIV-2infection against acute HIV-1 infection in PBM cells versus toxicity inPBM cells versus toxicity versus toxicity in macrophages * Calculatedusing MTT assay * Calculated using Propidium * Calculated using MTTassay Compound toxicity values Iodide toxicity values toxicity valuesJakafi >100 >100 >100 Tofacitinib 18.5 >50 >100 AZT >100 >50 >100

Example 20: Viability of Primary Human Lymphocytes Exposed to VariousConcentrations of Jakafi or Tofacitinib

The viability of primary human lymphocytes exposed to variousconcentrations of Jakafi or Tofacitinib was determined using thetechniques discussed above.

PHA and interleukin-2 (TL-2) stimulated primary human lymphocytes wereexposed to various concentrations of Jakafi or Tofacitinib for 5 daysprior to assessment of viability using propidium Iodide (flowcytometry).

FIGS. 4a-f show the results of flow cytometric analysis of PHA+IL-2stimulated primary human lymphocytes exposed to various concentrationsof Jakafi or Tofacitinib for 5 days prior to assessment of viabilityusing propidium iodide (flow cytometry).

A gating strategy based on forward scatter (FSC) and side scatter (SSC)was established, and used uniformly across all samples. FIG. 4a is ascatter plot showing a Side Scatter (SSC) gating strategy, where theX-axis in the first chart is Side Scatter Pulse Height (SSC-h) and theY-axis is Side Scatter Pulse Width (SSC-w), and in the second chart, theforward-scattered light (FSC) is shown with the X axis being ForwardScatter Pulse Height (FSC-H) and the Y axis being Forward Scatter PulseWidth (FSC-W). The gating strategy based on forward scatter (FSC) andside scatter (SSC) was established and used uniformly across allsamples.

Cells incubated in the absence of drug were 92.8% viable, and cellsexposed to 95° C. heat for 1 minute (positive control for dead cells)were 2.8% viable (FIG. 4b ).

Gating was established based on viable cells cultured in the absence ofdrug (FIG. 4b ). Histograms and scatter plots are representative datafrom at least 3 independent experiments conducted with pooled cells from8 donors. Graphs (E, F) are mean and standard deviations compiled fromeach independent experiment.

FIG. 4B is a histogram showing the results of flow cytometry studiesusing Propidium Iodide stain quantified with the phycoerythrin (PE-A)channel. Only dead/dying cells will stain positive for Propidium Iodide,therefore only dead/dying cells will be detected by the PE channel usingflow cytometry. Living, viable cells will not be stained by PropidiumIodide, therefore they will not be detected in the PE channel. Cellsincubated in the absence of drug were 92.8% viable (meaning that 92.8%of cells did not uptake Propidium Iodide stain), and the positivecontrol of cells exposed to 95° C. heat for 1 minute were 2.8% viable(meaning that 97.2% of these cells stained positive for Propidium Iodideand are therefore dead) (B). The data is shown in terms of total percentof cells in each sample, where gating was established based on viablecells cultured in the absence of drug.

FIG. 4c shows histograms comparing the cell viability for cells exposedto Jakafi and to no drug (i.e., controls) for concentrations of 0.1 μMJakafi, 1.0 μM Jakafi, 10 μM Jakafi, and 50 μM Jakafi.

FIG. 4d shows histograms comparing the cell viability for cells exposedto Tofacitinib and to no drug (i.e., controls) for concentrations of 0.1μM Tofacitinib, 1.0 μM Tofacitinib, 10 μM Tofacitinib, and 50 μMTofacitinib.

FIGS. 4e and 4f are charts showing the mean and standard deviations fromthe experiments shown in FIGS. 4c (Jakafi) and 4 d (Tofacitinib),respectively.

The data showed that Jakafi did not significantly reduce viabilityversus no drug controls for all concentrations tested with the exceptionof 50 μM (p<0.05)(FIG. 4c ). The data also showed that Tofacitinib didnot significantly reduce viability versus no drug controls for allconcentrations tested (FIG. 4d ).

Example 21: Viability of Primary Human Lymphocytes Exposed to VariousConcentrations of Jakafi or Tofacitinib

The antiviral potency of Jakafi and Tofacitinib was evaluated in primaryrhesus macaque lymphocytes and macrophages using the techniques discussabove. The antiviral potency was approximately 0.4 μM (EC₅₀) and 4.0 μM(EC₉₀) for both Jakafi and Tofacitinib in primary rhesus macaquemacrophages. The antiviral potency was 0.09±0.1 (EC₅₀) and 1.3±0.8(EC₉₀) in primary rhesus macaque macrophages. An AZT controldemonstrated antiviral potency as expected. The data (Shown in Table 5below) are mean and standard deviations from at least three independentexperiments.

TABLE 5 Acute Acute Acute Acute infection infection infection infectionin rhesus in rhesus in rhesus in rhesus macaque macaque macaque macaquemacrophages macrophages lymphocytes lymphocytes Drug EC₅₀(μM) EC₉₀ (μM)EC₅₀ (μM) EC₉₀ (μM) Jakafi 0.4 ± 0.2 4.2 ± 1.3 0.09 ± 0.1  1.3 ± 0.8Tofacitinib 0.3 ± 0.2 3.1 ± 0.9 0.3 ± 0.1 2.9 ± 0.5 AZT 0.08 ± 0.1  0.9± 0.7 0.002 ± 0.001 0.03 0.02

Example 22: Synergistic Antiviral Potency for Co-Administration ofJakafi and Tofacitinib in Primary Human Lymphocytes and Macrophages

The synergistic antiviral potentency for co-administration of Jakafi andTofacitinib was evaluated in primary human lymphocytes and macrophages,using the techniques described above.

Co-administration of Jakafi and Tofacitinib at a ratio of 1:4(lymphocytes) or 1:1 (macrophages) demonstrated synergistic antiviralpotency, as calculated by CalcuSyn (Biosoft, Inc., Cambridge, GreatBritain). The results are shown in FIGS. 5a and 5b . EC₅₀ and EC₉₀ inlymphocytes were decreased by 5-fold and 117-fold, respectively (dottedlines, FIG. 5a ). EC₅₀ and EC90 were markedly decreased in macrophages(FIG. 5b ).

Example 23: Antiviral Potency and of Jakafi and Tofacitinib AgainstVarious NRTI-Resistant HIV-1 in Primary Human Lymphocytes

The antiviral potency of Jakafi and Tofacitinib against variousNRTI-resistant HIV-1 was evaluated in primary human lymphocytes usingthe techniques described above.

The antiviral potency of Jakafi and Tofacitinib was not significantlydifferent for wild-type HIV-1xxLAI versus that of HIV-1 containingmutations K65R, M184V, L74V, A62V/V75I/F77LF116Y/Q151M, or 4xAZT(D67N/K70R/T215Y/K219Q). Various controls for each mutation demonstratedpotency or resistance as expected. Efavirenz (EFV) was similarly potentacross all NRTI resistant strains as expected. Data are mean andstandard deviations calculated from at least 4 independent experiments,with pooled cells from 8 donors and duplicates in each experiment.

The EC₅₀ data is shown in Table 6, and the EC₉₀ data is shown in Table7.

TABLE 6 (EC₅₀) AZT (−)FTC 3TC Tofacitinib Jakafi xxLAI  0.03 ± 0007 0.09± 0.02 0.8 ± 0.4 2.6 ± 1.3 0.3 ± 0.3 M184V 0.01 ± 0.02 10.1 ± 7.3  >101.6 ± 0.7 0.3 ± 0.3 K65R 0.04 ± 0.02 0.5 ± 0.4 2.5 ± 3.0 1.8 ± 0.8 0.2 ±0.3 L74V 0.02 ± 0.02 0.2 ± 0.2 0.6 ± 0.8 0.9 ± 1.0 0.1 ± 0.2A62V/V75I/F77L/ 4.6 ± 7.7 0.4 ± 0.3 0.5 ± 0.7 0.2 ± 0.2 0.03 ± 0.02F116Y/Q151M 4xAZT 0.1 ± 0.1 0.2 ± 0.1 0.7 ± 0.8 0.3 ± 0.2 0.09 ± 0.1 (D67N/K70R/ T215Y/K219Q) D4T ddI EFV TDF xxLAI 1.0 ± 0.5 11.5 ± 6.6 0.02 ± 0.03 0.2 ± 0.2 M184V 0.6 ± 0.8 11.5 ± 9.1   0.01 ± 0.006 0.09 ±0.03 K65R 1.5 ± 0.6 21.2 ± 18.3 0.007 0.4 ± 0.1 L74V 0.9 ± 0.8 13.2 ±8.5  0.06 ± 0.07 0.2 ± 0.1 A62V/V75I/F77L/  6.8 ± 05.7 40.5 ± 52.1 0.2 ±0.3 0.7 ± 0.8 F116Y/Q151M 4xAZT 27.8 ± 37.1 35.5 ± 31.0 0.07 ± 0.04 0.07± 0.04 (D67N/K70R/ T215Y/K219Q)

TABLE 7 (EC₉₀) AZT (−)FTC 3TC Tofacitinib Jakafi xxLAI  0.1 ± 0.08 0.8 ±0.4 3.1 ± 1.2 28.4 ± 16.7 6.1 ± 7.6 M184V 0.02 ± 0.01 41.3 ± 29.3 >1027.1 ± 15.7 3.2 ± 2.3 K65R 0.3 ± 0.1 2.4 ± 1.4 6.0 ± 5.3 81.2 ± 26.7 8.5± 8.1 L74V 0.2 ± 0.1 1.3 ± 1.0 2.9 ± 2.9 47.7 ± 45.3 3.2 ± 2.6A62V/V75I/F77L/ 41.2 ± 50.2 2.1 ± 1.6 2.7 ± 1.7 8.9 ± 8.8 1.5 ± 1.5F116Y/Q151M 4xAZT 53.3 ± 66.1 1.2 ± 0.1 3.4 ± 1.1 17.1 ± 4.5  2.4 ± 2.0(D67N/K70R/ T215Y/K219Q) D4T ddI EFV TDF xxLAI 6.4 ± 0.4 55.4 ± 23.0 0.2± 0.3 0.9 ± 0.8 M184V 2.6 ± 2.5 44.9 ± 26.2 0.08 ± 0.08 0.5 ± 0.3 K65R7.9 ± 0.3 86.7 ± 0.8  0.02 ± 0.01 1.6 ± 0.5 L74V 9.8 ± 2.4 80.9 ± 16.60.2 ± 0.3 0.2 ± 0.1 A62V/V75I/F77L/ 70.3 ± 51.4 83.5 ± 28.6 0.44 ± 0.6 3.6 ± 2.2 F116Y/Q151M 4xAZT 53.2 ± 66.3 77.1 ± 32.4 0.2 ± 0.2 1.2 ± 1.1(D67N/K70R/ T215Y/K219Q)

Example 24: Fold Increase 50 (FI₅₀) and Fold Increase 90 (FI₉₀) forJakafi and Tofacitinib Against Various NRTI-Resistant HIV-1 in PrimaryHuman Lymphocytes

The fold increase 50 (FI₅₀) and fold increase 90 (FI₉₀) for Jakafi andTofacitinib against various NRTI-resistant HIV-1 was evaluated inprimary human lymphocytes, using the techniques described above. FI₅₀ isthe ratio of EC₅₀ against mutant virus:EC₅₀ against wild-type xxLAI. 1₉₀is the ratio of EC₉₀ against mutant virus:EC₉₀ against wild-type xxLAI.There was not significant increase in F1₅₀ or FI₉₀ for Jakafi orTofacitinib treated cells. Controls of AZT, (+)FTC, 3TC, d4T, ddI,Efavirenz (EFV), TDF demonstrated sensitivity or resistance as expected.

The data is shown below in Table 8 (F₅₀) and Table 9 (F₉₀)

TABLE 8 (FI₅₀) AZT (−)FTC 3TC Tofacitinib Jakafi M184V 0.5 117 13.3 0.61.3 K65R 1.5 5.5 3.3 0.7 0.8 L74V 0.8 2 0.7 0.4 0.5 A62V/V75I/F77L/ 1844.2 0.6 0.1 0.1 F116Y/Q151M 4xAZT 5.2 2 0.9 0.1 0.3 (D67N/K70R/T215Y/K219Q) D4T ddI EFV TDF M184V 0.6 1 0.5 0.6 K65R 1.5 1.8 0.3 2.2L74V 0.9 1.1 2.6 1.4 A62V/V75I/F77L/ 6.9 3.5 7.7 3.9 F116Y/Q151M 4xAZT28.1 3.1 3.1 1.3 (D67N/K70R/ T215Y/K219Q)

TABLE 9 (FI₉₀) AZT (−)FTC 3TC Tofacitinib Jakafi M184V 0.13 50 3.2 1.00.5 K65R 1.5 2.9 1.9 2.9 1.4 L74V 0.9 1.6 0.9 1.7 0.5 A62V/V75I/F77L/242 2.5 0.9 0.3 0.3 F116Y/Q151M 4xAZT 314 1.4 1.1 0.61 0.4 (D67N/K70R/T215Y/K219Q) D4T ddI EFV TDF M184V 0.4 0.8 0.4 0.6 K65R 1.2 1.6 0.1 1.7L74V 1.5 1.1 1.1 1.2 A62V/V75I/F77L/ 11 1.5 2 3.9 F116Y/Q151M 4xAZT 8.31.4 1 1.3 (D67N/K70R/ T215Y/K219Q)

The data is also shown in FIGS. 6a and 6b . Jakafi and Tofacitinib didnot display a significant difference in FI₅₀ (FIG. 6a ) or FI₉₀ (FIG. 6b) versus wild type HIV-1xxLAI for HIV-1 containing M184V, K65R, L74V,A62V/V75/F77L/F116Y/Q151M, or 4xAZT (D67N/K70R/T215Y/K219Q) containingmutations.

Example 25: Effect of Various Jak Inhibitors on Proliferation andViability of PHA or PHA+IL-2 Stimulated Primary Human Lymphocytes

The effect of various Jak inhibitors on proliferation and viability ofPHA or PHA+IL-2 stimulated primary human lymphocytes was evaluated usingthe techniques described above. For PHA stimulated lymphocytes,viability and proliferation were not significantly different than thatof cell exposed to media alone for all concentrations of either Jakafior Tofacitinib (FIG. 7a and FIG. 7c ). For PHA+IL-2 stimulatedlymphocytes, viability was not significantly different than that ofcells exposed to media alone for all concentrations of either Jakafi orTofacitinib (FIG. 7b ), however proliferation was significantlyinhibited by 1.0 μM of Jakafi or Tofacitinib (FIG. 7d ).

For all experiments, cells were incubated with media alone ordrug-containing medium for 5 days prior to assessment of cell count andviability. Data are mean and standard deviations for at least threeindependent experiments conducted with at least 4 pooled donors, andduplicates within each experiment. The dotted bar represents mean cellcount or viability for cells maintained in drug-free medium.

Example 26: Tofacitinib and Jakafi Inhibit Reactivation of Latent HIV-1

The ability of Tofacitinib and Jakafi to inhibit reactivation of latentHIV-1 was evaluated using the techniques described in Bosque andPlanelles (2009) Induction of HIV-1 latency and reactivation in primarymemory CD4+ T cells; Blood 113: 58-65, and Jordan et al, (2003) HIVreproducibly establishes a latent infection after acute infection of Tcells in vitro; The EMBO Journal, Vol. 22 No. 8 pp. 1868±1877.Tofacitinib (diamonds) and Jakafi (squares) inhibit reactivation oflatent HIV-1 in a primary central memory-based T cell latency model(FIG. 8a ) and in the J-Lat latency T cell system (FIG. 8b ). Jakafi wasthe more potent inhibitor across both systems, and inhibited Z 50% ofreactivation at concentrations found during steady-state or C_(max) invivo (shaded boxes).

The ability of Tofacitinib and Jakafi to inhibit reactivation of latentHIV-1 was also evaluated in primary human macrophages. Primary humanmonocytes were obtained by elutriation and differentiated to terminallydifferentiated macrophages in the presence of m-CSF for 5 days. Cellswere subsequently infected with VSV-pseudotyped HIV-1 (envelope region),allowing for 100% infection rate of cultures. Cells were furthercultured for 40 days until cultures were no longer producing HIV-1. Atthis time, all macrophages are now resting, latently infected cells. 41days post infection, 10 ng/ml phorbol myristate acetate (PMA) wasapplied to the latently infected macrophages for 24 hr in either theabsence of drug (positive control), or in the presence of 1.0 or 10.0 μMJakafi or Tofacitinib. After 24 hr., both PMA and drug containingmediums were removed, and cells were cultured in media alone. Sampleswere taken at various days post reactivation and extracellular,reactivated virus production was quantified using p24 ELISA. Results arereported as percent inhibition of reactivation of latent HIV-1 versus nodrug control. The results are shown in FIGS. 9a (Tofacitinib) and 9 b(Jakafi). Both Tofacitinib and Jakafi inhibit reactivation of latentHIV-1 in primary human macrophages when drug is applied to cells duringreactivation, but removed thereafter. Tofacitinib inhibits ˜40% ofreactivation while Jakafi inhibits ˜35% of reactivation within 72 hrpost reactivation.

Example 27: Tofacitinib and Jakafi Inhibit a Pro-HIV Cytokine (IFN-α)Induced Activation of the Jak-STAT Pathway

Tofacitinib and Jakafi inhibit a pro-HIV cytokine (IFN-α) inducedactivation of the Jak-STAT pathway. Jakafi and Tofacitinib inhibit IFN-αinduced phosphorylation of STAT1, 3, and 5 in primary CD4+T lymphocytesat sub-micromolar concentrations (A, B, C).

Example 28: Tofacitinib and Jakafi Inhibit a Pro-HIV Cytokine (IFN-α)Induced Activation of the Jak-STAT Pathway

The ability of Tofacitinib and Jakafi to inhibit a pro-HIV cytokine(IFN-α) induced activation of the Jak-STAT pathway was evaluated usingtechniques described above. Jakafi and Tofacitinib inhibit IFN-α inducedphosphorylation of STAT1, 3, and 5 in primary CD4+T lymphocytes atsub-micromolar concentrations, as shown below in Table 10. Both drugsalso inhibit pSTAT1, 3, and 5 with similar EC_(50/90) in CD8 T cells andCD14 monocytes (data not shown).

TABLE 10 EC₅₀/₉₀ pSTAT1 EC₅₀/₉₀ pSTAT3 EC₅₀/₉₀ pSTAT5 Drug (μM) (μM)(μM) (Tofacitinib/ <0.01/0.01  0.02/0.9 <0.01/0.01  Xalijenz) (Jakafi)<0.01/<0.01 <0.01/0.01 <0.01/<0.01

While the foregoing specification teaches the principles of the presentinvention, with examples provided for the purpose of illustration, itwill be understood that the practice of the invention encompasses all ofthe usual variations, adaptations and/or modifications as come withinthe scope of the following claims and their equivalents. All referencescited herein are incorporated by reference in their entirety for allpurposes.

Example 29: Jak Inhibitors Tofacitinib and Ruxolitinib Reduce theFrequency of Cells Harboring Integrated Viral DNA and IL-15-InducedReactivation of Latent HIV-1 in CD4 T Cells (FIG. 11)

FIG. 11 shows that tofacitinib and ruxolitinib block reservoirreactivation and decay the size of the HIV reservoir in CD4 T cells fromHIV-infected individuals, both alone and in the presence of HAART.

Example 30: Jak Inhibitors Block Bystander Infection in Primary CD4 TCells

FIG. 12 shows that ruxolitinib blocks bystander CD4 T cells frombecoming infected by their neighbor cells, thereby preventing seedingand expansion of the HIV reservoir, and also preventing establishment ofthe HIV reservoir.

Example 31: Jak Inhibitor can Block the HIV Establishment, Maintenance,Lifespan, and HIV-Induced Inflammatory Events (HIV-AssociatedNeurocognitive Impairment System) in Primary Human Macrophages andMonocytes

FIG. 13 shows that baricitinib blocks HIV infection, HIV-inducedinflammation and activation, HIV reactivation, and decays the lifespanof the HIV reservoir in primary human macrophages and monocytes, whichare resident cells of the CNS. These data show that baricitinib blockskey events that drive HIV-associated neurocognitive dysfunction (HAND)in monocytes and macrophages.

1-25. (canceled)
 26. A method of treating, preventing, a Coronaviridae infection, comprising administering an effective, antiviral amount of a compound selected from the group consisting of Jakafi, Tofacitinib, and LY3009104/INCB28050 to a patient in need of treatment thereof.
 27. The method of claim 26, wherein the compound is LY3009104/INCB28050.
 28. The method of claim 26, wherein the Coronaviridiae infection is treated.
 29. The method of claim 26, wherein the Coronaviridiae infection is prevented.
 30. The method of claim 27, wherein the Coronaviridiae infection is treated.
 31. The method of claim 27, wherein the Coronaviridiae infection is prevented. 